Tricyclic Benzoxaborole Compounds and Uses Thereof

ABSTRACT

Compounds of Formula II, 
     
       
         
         
             
             
         
       
         
         wherein X is selected from chloro, fluoro, bromo and iodo, R 1  and R 2  are each independently selected from H, —CH 3 , —CH 2 CH 3 , —CH 2 CH 2 CH 3 , or —CH(CH 3 ) 2 ; compositions containing them, their use in therapy, including their use as anti-mycobacterial agents, for example in the treatment of a mycobacterial infection in a mammal, and methods for the preparation of such compounds, are provided.

PRIORITY

This application is a continuation of and claims priority to U.S.application Ser. No. 15/792,251 filed Oct. 24, 2017 which is acontinuation of and claims priority to U.S. application Ser. No.14/387,384 filed Sep. 23, 2014 pursuant to 35 U.S.C. § 371 as a UnitedStates National Phase Application of International Patent ApplicationSerial No. PCT/US2014/050370 filed Aug. 8, 2014, which claims priorityto U.S. Provisional Application No. 61/864,496 filed Aug. 9, 2013, andU.S. Provisional Application No. 61/918,976 filed Dec. 20, 2013, and theentire contents of each of the foregoing applications are herebyincorporated by reference.

FIELD OF THE INVENTION

This invention relates to compounds, compositions containing them, theiruse in therapy, including their use as anti-mycobacterials, for examplein the treatment of tuberculosis, and methods for the preparation ofsuch compounds.

BACKGROUND OF THE INVENTION

Mycobacterium is a genus in the class of bacteria called Actinobacteriawith its own distinct family known as Mycobacteriacae. Mycobacteriumcontains various obligate and opportunistic pathogens of animals, whichmay also be transmitted to humans and cause disease in humans, thusexhibiting a considerable zoonotic potential. During the past fewdecades, members of the Mycobacterium avium-intracellulare complex(MAIC) emerged as pathogens of human diseases, including lymphadenitisin children, pulmonary tuberculosis-like disease, and disseminatedinfections (occurring predominantly in immunocompromised persons,particularly AIDS patients). Similarly, important animal diseases resultfrom infections in an animal by members of this group, e.g., aviantuberculosis and paratuberculosis in ruminants. MAIC includes M.intracellulare and 4 subspecies of M. avium, namely, M. avium subsp.avium, M. avium subsp. hominissuis, M. avium subsp. silvaticum, and M.avium subsp. paratuberculosis. Whereas members of the M. tuberculosiscomplex are transmitted by direct host contact, MAIC species areacquired predominantly from environmental sources, including soil,water, dust, and feed.

Mycobacterium tuberculosis (MTB) is a small aerobic non-motile high-GCbacillus with an “outer-membrane” that is unusually thick, “waxy,”hydrophobic, rich in mycolic acids, and extremely impermeable, makingmycobacterium infections difficult to treat. One third of the world'spopulation is thought to be infected (including latent MTB), but thisnumber increases to upwards of 80% of the population in many Asian andAfrican countries. If untreated, the death rate from active MTBinfections is more than 50%. In addition, the combination of HIV and MTBis deadly and increasing numbers of MTB strains are becoming resistantto standard of care drugs; approximately 300,000 new cases of multidrugresistant (MDR) M. tuberculosis are reported each year. Multidrugresistant (MDR) M. tuberculosis are resistant to isoniazid andrifampicin, and extensive drug resistant (XDR) M. tuberculosis are alsoresistant to at least one quinolone and one aminoglycoside. As can beseen in FIG. 1, XDR M. tuberculosis has been reported across much of theglobe.

Add to these issues the ease of transmission, as shown in FIG. 2, theglobalization of travel, and the ongoing relocation and emigration ofmany segments of the world's population and it is apparent that MTB isbecoming a global crisis.

Synthetic drugs for treating tuberculosis (TB) have been available forover half a century, but incidences of the disease continue to riseworld-wide. More than 2 billion people are currently infected with M.tuberculosis, most being latent cases, and it is estimated that over 9million new cases occur each year, worldwide, resulting in from 1.7 tonearly 2 million deaths per year. In 2004 alone approximately 24,500 newinfections and close to 5,500 deaths were recorded, each day. SeeZignol, Met al., M. Surveillance of anti-tuberculosis drug resistance inthe world: an updated analysis, 2007-2010. Bull. World Health Organ2012, 90 (2), 111-119D) Co-infection with HIV is driving the increase inincidence (Williams, B. G.; Dye, C. Science, 2003, 301, 1535) and thecause of death in 31% of AIDS patients in Africa can be attributed toTB. See Corbett, E. Let al.,. Arch. Intl. Med., 2003, 163, 1009,Septkowitz, Aet al., Clin. Microbiol. Rev. 1995, 8, 180).

The limitations of tuberculosis therapy and prevention are well known.The current available vaccine, BCG was introduced in 1921 and fails toprotect most people past childhood. According to a 2006report—“International Standards for Tuberculosis Care”, a documentdeveloped by the Tuberculosis Coalition for Technical Assistance (TBCTA)which partners include Centers for Disease Control, American ThoracicSociety, Tuberculosis Foundation, KNCV, the World Health Organizationand the International Union Against Tuberculosis and LungDisease—patients who do become infected with active disease currentlyendure two months of combination therapy with medicines introducedbetween 50 and 60 years ago—isoniazid (1952), rifampin (1963),pyrazinamide (1954) and ethambutol (1961)—followed by another 4 monthsof isoniazid and rifampin (also known as rifampicin). Alternatively thecontinuation phase could include Isoniazid and ethambutol for six monthswhen adherence cannot be assessed, but according to this report, alonger continuation phase is associated with a higher rate of failureand relapse, especially in patients with HIV infection. Moreover, asdetailed in this report, the doses of antituberculosis drugs used shouldconform to international recommendation and fixed-dose combinations oftwo (isoniazid and rifampicin), three (isoniazid, rifampicin, andpyrazinamide), and four (isoniazid, rifampicin, pyrazinamide, andethambutol) drugs are highly recommended, especially when it is notpossible to monitor the patient to ensure the treatment is ingested.

Daily dosing is required in these treatment phases and poor compliancedrives the emergence and spread of multi-drug-resistant strains, whichare challenging to treat. Shorter courses of more active agents whichcan be taken less frequently and which present a high barrier to theemergence of resistance, i.e. agents which are effective againstmulti-drug resistant strains of TB (MDR-TB), are urgently required. AMarch 2013 report(http://www.aidsmap.com/Once-weekly-continuation-phase-TB-treatment-equals-standard-of-care/page/2589498/)suggests that a two-drug combination of rifapentine (a long-actingderivative of rifampicin) with moxifloxacin (a fluoroquinoloneantibiotic that has not been used previously in TB treatment) can allowtuberculosis (TB) treatment to be taken once-weekly during thefour-month continuation phase and achieves the same standard of care asthe traditional continuation treatment of daily treatment with isoniazidand rifampin. Such a treatment phase would allow treatment supervisionto extend throughout the continuation phase, increasing adherence.However, moxifloxacin is not yet approved for treatment of TB, and theonce-weekly treatment protocol is not yet endorsed or approved as analternative standard of care treatment—guideline panels at internationaland national levels will need to review the published evidence todetermine if this alternative continuation treatment protocol should berecommended and adopted. In addition, rifapentine is expensive, andinteractions between rifapentine and antiretroviral drugs in thenon-nucleoside reverse transcriptase inhibitor (NNRTI) and proteaseinhibitor classes may prevent its use in TB patients who are also HIVpositive and taking antiretroviral medicines. Thus, at present, thecosts/benefits analysis of a continuation treatment with weeklyrifapentine versus daily rifampicin is yet to be fully assessed.

The tuberculosis drug Sirturo™ (bedaquiline) was approved in the UnitedStates in late December 2012, and another, delamanid, is attempting togain regulatory approval in the EU. However, both are reserved fordrug-resistant tuberculosis, which accounts for just 5% of new cases. A2007 Editorial and News Focus in Nature Medicine discusses many aspectsof TB such as pathogenesis, epidemiology, drug discovery and vaccinedevelopment to date (Nature Medicine, 2007, Focus on Tuberculosis, Vol13(3), pages 263-312), noting that 125 years after the anniversary ofthe discovery of Mycobacterium tuberculosis, more than one-third ofpeople in the world are infected with M. tuberculosis, and of these,more than 1 in 10 will develop the disease known as tuberculosis,formerly known as consumption, in their lifetime.

When coupled with the emergence of multi-drug resistant strains ofMycobacterium tuberculosis (MDR-TB), the scale of the problem isamplified. The global rise of bacteria and other microorganismsresistant to antibiotics and antimicrobials in general, poses a majorthreat. Deployment of massive quantities of antimicrobial agents intothe ecosphere during the past 60 years has introduced a powerfulselective pressure for the emergence and spread ofantimicrobial-resistant pathogens. There is therefore a need to discoverand develop new chemical entities to treat TB (recent leads are reviewedin: Grosset J H, Singer T G, Bishai W R. New Drugs for the Treatment ofTuberculosis: Hope and Reality. Int J Tuberc Lung Dis. 2012 August;16(8):1005-14).

The present invention relates to tricyclic benzoxaborole compounds thatshow unexpected selectivity for inhibiting replication of Mycobacteriumtuberculosis (M. tuberculosis) versus inhibition (toxicity) of humancells compared to other benzoxaborole compounds, and exhibitsub-micromolar MIC values against mycobacterium species, particularlyMycobacterium tuberculosis and Mycobacterium tuberculosis complex (MTC),Mycobacterium avium and Mycobacterium avium complex (MAC) andMycobacterium avium intracellulare complex (MAIC). Generally speaking, abenzoxaborole has the following structure and substituent numberingsystem:

Certain benzoxaboroles which are substituted at position 7 form atricyclic benzoxaborole compound. When the resulting tricyclicbenzoxaborole is additionally substituted with a halogen substituent atposition 4 and an aminomethyl substituent at position 3, such compoundsare surprisingly selective towards and effective against mycobacteriaincluding M. tuberculosis. The selectivity observed is assessed bycomparing MIC values for such compounds relative to inhibition(toxicity) of these compounds to human cells, compared to otherbenzoxaborole compounds.

Boron-containing molecules such as benzoxaboroles that are useful asantimicrobials have been described previously, see e.g.“Benzoxaboroles—Old compounds with new applications” Adamczyk-Woźniak,A. et al., Journal of Organometallic Chemistry Volume 694, Issue 22, 15Oct. 2009, Pages 3533-3541, and U.S. Pat. Pubs. US20060234981,US20070155699, US20090227541, WO2012033858, and US2013165411.

US20090227541 discloses a multitude of compounds, including twotricyclic benzoxaborole compounds with differing antibacterial activityagainst a panel of Gram negative bacteria (See e.g. Tables 1 and 2), butdoes not disclose tricyclic benzoxaborole compounds with halogensubstitution on the benzoxaborole ring. WO2012033858 disclosesbenzoxaborole compounds with activity against Mycobacteriumtuberculosis, including certain benzoxaborole compounds (see e.g.Examples 1.A through 1.V), but again, no tricyclic benzoxaborolecompounds are disclosed with halogen substitution on the benzoxaborolering. US2013165411 discloses tricyclic benzoxaborole compounds showingactivity against Acinetobacter baumannii, Pseudomonas aeruginosa,Escherichia coli and Klebsiella pneumoniae (see Table 1), but notesspecifically that the halogen-substituted tricyclic compoundsinvestigated (Examples 17, 18 and 19) lack activity against A.baumannii, with MIC values ≥16 μg/μL antibacterial activity (see FIG.1B).

SUMMARY OF THE INVENTION

The inventors have surprisingly found that tricyclic benzoxaborolecompounds as described herein show unexpected selectivity for inhibitingreplication of Mycobacterium tuberculosis (M. tuberculosis) versusinhibition (toxicity) of human cells compared to other benzoxaborolecompounds. These tricyclic benzoxaborole compounds exhibitsub-micromolar MIC values against M. tuberculosis, which is comparableto or better than the MIC values for current therapies available forinhibiting M. tuberculosis. Further, in other embodiments, the tricyclicbenzoxaborole compounds as described herein are envisioned for use incombination with current anti-tubercular compounds and are envisioned toachieve greater efficacy in treating animals, including humans, infectedwith M. tuberculosis.

Resistance remains an issue in the treatment of tuberculosis (TB) andone clinical strategy is to focus on early combination with other TBdrugs and to expedite early assessment of the compound's efficacy inpatients. Compounds of Formula II or Formula IIa offer a uniqueopportunity to address the serious issues which arise during thetreatment of TB, such as multi-drug resistance, extensive-drugresistance, reactivity and/or adverse interaction between therapeuticagents in a multi-drug combination, and treatment length, therebyaddressing potential patient needs.

In certain embodiments of the present invention there is featuredcombinations of anti-tuberculosis agents and certain tricyclicbenzoxaboroles, for use in the treatment of Mycobacterium tuberculosisinfections in animals, including humans. In particular embodiments, suchtricyclic benzoxaboroles are used, in combination with other knowanti-tuberculosis agents, for treating an animal subject with aMycobacterium tuberculosis infection, particularly in an animal subjectthat is additionally infected with a human retrovirus, in particular ahuman immunodeficiency virus (HIV).

In an exemplary embodiment, the invention is a compound as describedherein, or a pharmaceutically acceptable salt thereof.

In particular embodiments, the tricyclic benzoxaborole is a compound ora salt thereof, including a pharmaceutically acceptable salt thereof,having a structure according to Formula II:

wherein X is selected from chloro, fluoro, bromo and iodo; R¹ and R² areeach independently selected from H, —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, and—CH(CH₃)₂,.

In particular embodiments there is provided a compound of Formula II ora salt thereof, wherein X is chloro or bromo; R¹ and R² are eachindependently selected from H, —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, and —CH(CH₃)₂.

In particular embodiments there is provided a compound of Formula II ora salt thereof, wherein X is fluoro, R¹ and R² are as described herein.

In particular embodiments there is provided a compound of Formula II ora salt thereof, wherein X is chloro, R¹ and R² are as described herein.

In particular embodiments there is provided a compound of Formula II ora salt thereof, wherein X is bromo, R¹ and R² are as described herein.

In particular embodiments there is provided a compound of Formula II ora salt thereof, wherein X is iodo, R¹ and R² are as described herein.

In particular embodiments there is provided a compound of Formula II ora salt thereof, wherein X is chloro or bromo, R¹ and R² are eachindependently selected from H, —CH₃, and —CH₂CH₃.

In particular embodiments there is provided a compound of Formula II ora salt thereof, wherein X is chloro or bromo, R¹ and R² are eachindependently selected from H and —CH₃.

In particular embodiments there is provided a compound of Formula II ora salt thereof, wherein X is fluoro or iodo, R¹ and R² are eachindependently selected from H and —CH₃.

In particular embodiments there is provided a compound of Formula IIa

wherein X is fluoro, chloro, bromo or iodo, and R¹ and R² are eachindependently selected from H, —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, and —CH(CH₃)₂,or a salt thereof, including a pharmaceutically acceptable salt thereof.

In particular embodiments there is provided a compound of Formula IIawherein X is fluoro, chloro, bromo or iodo and R¹ and R² are eachindependently selected from H, —CH₃, and —CH₂CH₃, or a salt thereof,including a pharmaceutically acceptable salt thereof.

In particular embodiments there is provided a compound of Formula IIawherein X is fluoro, chloro, bromo or iodo and R¹ and R² are eachindependently selected from H and —CH₃, or a salt thereof, including apharmaceutically acceptable salt thereof.

In particular embodiments there is provided a compound of Formula IIa ora salt thereof, wherein X is fluoro, and R¹ and R² are as describedherein.

In particular embodiments there is provided a compound of Formula IIa ora salt thereof, wherein X is chloro, and R¹ and R² are as describedherein.

In particular embodiments there is provided a compound of Formula IIa ora salt thereof, wherein X is bromo, and R¹ and R² are as describedherein.

In particular embodiments there is provided a compound of Formula IIa ora salt thereof, wherein X is iodo, and R¹ and R² are as describedherein.

In particular embodiments there is provided a compound of Formula IIawherein X is chloro or bromo and R¹ and R² are each independentlyselected from H, —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, and —CH(CH₃)₂, or a saltthereof, including a pharmaceutically acceptable salt thereof.

In particular embodiments there is provided a compound of Formula IIawherein X is chloro or bromo, and R¹ and R² are each independentlyselected from H, —CH₃, and —CH₂CH₃, or a salt thereof, including apharmaceutically acceptable salt thereof.

In particular embodiments there is provided a compound of Formula IIawherein X is chloro or bromo, and R¹ and R² are each independentlyselected from H and —CH₃, or a salt thereof, including apharmaceutically acceptable salt thereof.

In particular embodiments, the tricyclic benzoxaborole is a compound of

Formula II as indicated below:

or a pharmaceutically acceptable salt thereof.

In particular embodiments, the tricyclic benzoxaborole is a compound ofFormula IIa as indicated below:

or a pharmaceutically acceptable salt thereof.

In other embodiments, the tricyclic benzoxaborole is a compound ofFormula II as indicated below:

wherein X is as defined herein, or a pharmaceutically acceptable saltthereof.

In other embodiments, the tricyclic benzoxaborole is a compound ofFormula IIa as indicated below:

wherein X is as defined herein, or a pharmaceutically acceptable saltthereof.

In still other embodiments, the tricyclic benzoxaborole is a compound ofFormula II as indicated below:

and a pharmaceutically acceptable salt thereof.

In still other embodiments, the tricyclic benzoxaborole is a compound ofFormula IIa as indicated below:

or a pharmaceutically acceptable salt thereof.

In another embodiment there is provided a compound,(S)-(3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine,having the formula:

In another embodiment there is provided a compound,(S)-(3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine,having the formula:

or a pharmaceutically acceptable salt thereof.

Another embodiments provides a pharmaceutically acceptable salt of acompound, (S)-(3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine, having the formula:

Another embodiment provides a pharmaceutical composition comprising acompound,(S)-(3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine,having the formula:

together with at least one pharmaceutically acceptable excipient.

In yet another embodiment there is provided a compound,(S)-(3-bromo-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine,having the formula:

Still another embodiment provides a compound,(S)-(3-bromo-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine,having the formula:

or a pharmaceutically acceptable salt thereof.

Another embodiment provides a pharmaceutically acceptable salt of acompound,(S)-(3-bromo-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine,having the formula:

Another embodiment provides a pharmaceutical composition comprising acompound,(S)-(3-bromo-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine,having the formula:

together with at least one pharmaceutically acceptable excipient.

One embodiment provides a compound of Formula II or Formula IIa or asalt thereof, which is:

-   -   (3-bromo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;    -   (S)-(3-bromo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;    -   (3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;    -   (S)-(3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;    -   (3-chloro-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;    -   (3-bromo-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;    -   (3-bromo-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;    -   (S)-(3-bromo-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;    -   (3-chloro-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;    -   (S)-(3-chloro-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;    -   (3-fluoro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;        or    -   (S)-(3-iodo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine.

In a related embodiment, the pharmaceutically acceptable salt isselected from hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like. In other related embodiments, thepharmaceutically acceptable salt is derived from organic acids includingacetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid,benzoic acid, succinic acid, suberic acid, fumaric acid, glucaronicacid, galacturonic acid, lactic acid, mandelic acid, phthalic acid,benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid,methanesulfonic acid, and the like. Still other related embodiments thepharmaceutically acceptable salt includes salts of amino acids such asarginate, lysinate and the like.

In particular aspects of the invention, the compound of Formula II orFormula IIa is a mixture of diastereomers. In other particular aspectsof the invention, the compound of Formula II or Formula IIa is adiastereomer. In other particular aspects of the invention, the compoundof Formula II is a racemic mixture of enantiomers. In still otherparticular aspects of the invention, the compound of Formula II is aspecific enantiomer. In particular aspects of the invention when R¹ andR² are both H or CH₃, the compound of Formula II or Formula IIa has (S)stereochemistry at the chiral center. One embodiment provides acombination comprising: a first therapeutic agent wherein the firsttherapeutic agent is a compound as described herein, or apharmaceutically acceptable salt thereof; optionally a secondtherapeutic agent; optionally a third therapeutic agent; optionally afourth therapeutic agent; optionally a fifth therapeutic agent; andoptionally a sixth therapeutic agent.

A related embodiment provides a combination as described wherein theoptional second, third, fourth, fifth and sixth therapeutic agent isindependently selected from isoniazid, rifampin, pyrazinamide,ethambutol, moxifloxacin, rifapentine, clofazimine, bedaquiline(TMC207), nitroimidazo-oxazine PA-824, delamanid (OPC-67683), anoxazolidinone such as linezolid, tedizolid, radezolid, sutezolid(PNU-100480), or posizolid (AZD-5847), EMB analogue SQ109, abenzothiazinone, a dinitrobenzamide or an antiviral agent including anantiretroviral agent.

A related embodiment provides a combination as described wherein theantiretroviral agents is zidovudine, didanosine, lamivudine,zalcitabine, abacavir, stavudine, adefovir, adefovir dipivoxil,fozivudine, todoxil, emtricitabine, alovudine, amdoxovir, elvucitabine,nevirapine, delavirdine, efavirenz, loviride, immunocal, oltipraz,capravirine, lersivirine, GSK2248761, TMC-278, TMC-125, etravirine,saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, fosamprenavir,brecanavir, darunavir, atazanavir, tipranavir, palinavir, lasinavir,enfuvirtide, T-20, T-1249, PRO-542, PRO-140, TNX-355, BMS-806,BMS-663068 and BMS-626529, 5-Helix, raltegravir, elvitegravir,GSK1349572, GSK1265744, vicriviroc (Sch-C), Sch-D, TAK779, maraviroc,TAK449, didanosine, tenofovir, lopinavir, or darunavir.

Another embodiment of the invention provides a combination as describedwherein the second, third, fourth, fifth and sixth therapeutic agent isselected from a therapeutic agent approved or recommended for thetreatment of tuberculosis.

One embodiment of the present invention provides a pharmaceuticalformulation comprising a first therapeutic agent, said first therapeuticagent being a therapeutically effective amount of a compound of FormulaII or Formula IIa according to any of the embodiments described hereinor a pharmaceutically acceptable salt thereof. A related embodimentprovides a combination as described herein and a pharmaceuticallyacceptable excipient, adjuvant or diluent. In another embodiment, thepharmaceutical formulation may further comprise a second therapeuticagent.

Another embodiment provides a method of killing mycobacteria and/orinhibiting replication of mycobacteria that causes disease in an animal,comprising contacting the mycobacteria with an effective amount of acompound of Formula II or Formula IIa as described herein or apharmaceutically acceptable salt thereof, so as to kill the mycobacteriaand/or prevent the replication of the mycobacteria.

Another embodiment of the invention provides a method of treating amycobacterium infection in an animal comprising: administering to theanimal any one of: (i) a therapeutically effective amount of a compoundof Formula II or Formula IIa as described herein or a pharmaceuticallyacceptable salt thereof; (ii) a therapeutically effective amount of acombination comprising a compound of Formula II or Formula IIa asdescribed herein or a pharmaceutically acceptable salt thereof; or (iii)a therapeutically effective amount of a pharmaceutical formulationcomprising a compound of Formula II or Formula IIa as described hereinor a pharmaceutically acceptable salt thereof, so as to treat themycobacterium infection in the animal.

In a further aspect, the invention provides a method of killingmycobacteria and/or inhibiting replication of mycobactera or a method oftreating a mycobacterial infection in an animal such as livestock andpets, including cattle sheep, goats, dogs and cats, or a human,including an immune-suppressed human said method comprising: contactingthe mycobactera with an effective amount of a compound of Formula II orFormula IIa as described herein, thereby killing the mycobacteria and/orinhibiting replication of the mycobacteria, or said method comprisingadministering to the animal with the mycobacterial infection atherapeutically effective amount of a compound of Formula II or acompound of Formula IIa, or a pharmaceutically acceptable salt thereof.In an exemplary embodiment, the compound of Formula II or compound ofFormula IIa is part of a pharmaceutical formulation described herein. Inanother exemplary embodiment, the contacting occurs under conditionswhich permit entry of the combination into the mycobacterium.

Another embodiment of the invention provides a method as describedherein, wherein the mycobacteria is selected from Mycobacteriumtuberculosis, Mycobacterium avium including subspecies (subsp.)Mycobacterium avium subsp. avium, Mycobacterium avium subsp.hominissuis, Mycobacterium avium subsp. silvaticum, and Mycobacteriumavium subsp. paratuberculosis; Mycobacterium kansasii, Mycobacteriummalmoense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacteriumxenopi, Mycobacterium scrofulaceum, Mycobacterium abscessus,Mycobacterium chelonae, Mycobacterium haemophilum, Mycobacterium leprae,Mycobacterium marinum, Mycobacterium fortuitum, Mycobacteriumparafortuitum, Mycobacterium gordonae, Mycobacterium vaccae,Mycobacterium bovis, Mycobacterium bovis BCG, Mycobacterium africanum,Mycobacterium canetti, Mycobacterium caprae, Mycobacterium microti,Mycobacterium pinnipedi, Mycobacterium leprae, Mycobacterium ulcerans,Mycobacterium intracellulare, Mycobacterium tuberculosis complex. (MTC),Mycobacterium avium complex (MAC), Mycobacterium avian-intracellularecomplex (MAIC), Mycobacterium gordonae clade; Mycobacterium kansasiiclade; Mycobacterium chelonae clade; Mycobacterium fortuitum clade;Mycobacterium parafortuitum clade; and Mycobacterium vaccae clade.

Another embodiment provides a method of treating a mycobacteriuminfection in an animal comprising: administering to the animal any oneof: (i) a therapeutically effective amount of a compound of Formula IIor Formula IIa as described herein or a pharmaceutically acceptable saltthereof; (ii) a therapeutically effective amount of a combinationcomprising a compound of Formula II or Formula IIa as described hereinor a pharmaceutically acceptable salt thereof; or (iii) atherapeutically effective amount of a pharmaceutical formulationcomprising a compound of Formula II or Formula IIa as described hereinor a pharmaceutically acceptable salt thereof, so as to treat themycobacterium infection in the animal, wherein the mycobacteriuminfection is a M. tuberculosis infection.

Another embodiment provides a compound of Formula II or Formula IIa asdescribed herein or a pharmaceutically acceptable salt thereof, for usein the treatment of a disease resulting from a mycobacterial infectionin an animal, including a human. Another embodiment provides a compoundas described herein, wherein the disease is selected from tuberculosis,leprosy, Johne's disease, Buruli or Bairnsdale ulcer, Crohn's disease,pulmonary disease or pulmonary infection. pneumonia, bursa, synovial,tendon sheaths, localized abscess, lymphadenitis, skin and soft tissueinfections Lady Windermere syndrome, MAC lung disease, disseminatedMycobacterium avium complex (DMAC), disseminated Mycobacterium aviumintracellulare complex (DMAIC), hot-tub lung, MAC mastitis, MACpyomyositis, Mycobacterium avium paratuberculosis, or granuloma,disease.

One embodiment provides the use of a compound of Formula II or FormulaIIa as described herein or a pharmaceutically acceptable salt thereof inthe manufacture of a medicament for the treatment of mycobacterialinfection in an animal.

Another embodiment provides a method of treating a disease resultingfrom a mycobacterial infection in an animal, particularly in a mammal,more particularly in a human, which method comprises administering tothe animal in need of such treatment an effective amount of a compoundFormula II as described herein or a pharmaceutically acceptable saltthereof. Another embodiment provides a method as described, wherein thedisease is selected from tuberculosis, leprosy, Johne's disease, Burulior Bairnsdale ulcer, Crohn's disease, pulmonary disease or pulmonaryinfection. pneumonia, bursa, synovial, tendon sheaths, localizedabscess, lymphadenitis, skin and soft tissue infections Lady Windermeresyndrome, MAC lung disease, disseminated Mycobacterium avium complex(DMAC), disseminated Mycobacterium avium intracellulare complex (DMAIC),hot-tub lung, MAC mastitis, MAC pyomyositis, Mycobacterium aviumparatuberculosis, or granuloma disease.

Another embodiment provides a method of treating a mycobacterialinfection in an animal, particularly in a mammal, which method comprisesadministering to the animal in need of such treatment a therapeuticallyeffective amount of a compound described herein, or pharmaceuticallyacceptable salt thereof. Another embodiment provides a method oftreating a mycobacterial infection in an animal, particularly a mammal,wherein the mycobacterial infection is Mycobacterium tuberculosis.

In one embodiment there is provided a pharmaceutical formulationcomprising a first therapeutic agent, said first therapeutic agent beinga therapeutically effective amount of a compound described herein orpharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable excipient, adjuvant or diluent.

More particularly, a pharmaceutical formulation is provided comprising afirst therapeutic agent that is a compound of Formula II or Formula IIa,said first therapeutic agent being a therapeutically effective amount ofa compound as described herein or pharmaceutically acceptable saltthereof, in any embodiment as described herein; a pharmaceuticallyacceptable excipient, adjuvant or diluent; and a second therapeuticagent that is not a compound of Formula II or Formula IIa. In relatedaspects, the pharmaceutical formulation comprises a first therapeuticagent that is a compound of Formula II or Formula IIa as describedherein, or a pharmaceutically acceptable salt thereof, and optionallycomprises a second therapeutic agent that is not a compound of FormulaII or Formula IIa, and optionally comprises a third therapeutic agent,and optionally comprises a fourth therapeutic agent, and optionallycomprises a fifth therapeutic agent, and optionally comprises a sixththerapeutic agent. In related aspects, the second, third, fourth, fifthand sixth therapeutic agent is an anti-mycobacterial agent other than acompound of Formula II or Formula IIA. In related aspects, the second,third, fourth, fifth and sixth therapeutic agent is selected fromisoniazid, rifampin, pyrazinamide, ethambutol, moxifloxacin,rifapentine, clofazimine, bedaquiline (TMC207), nitroimidazo-oxazinePA-824, delamanid (OPC-67683), oxazolidinone such as linezolid,tedizolid, radezolid, sutezolid (PNU-100480), and posizolid (AZD-5847),EMB analogue SQ109, a benzothiazinone, a dinitrobenzamide and anantiviral agent including an antiretroviral agent. In related aspects,the second, third, fourth, fifth and sixth therapeutic agent is atherapeutic agent approved and/or recommended for the treatment oftuberculosis.

A related embodiment provides a pharmaceutical formulation comprising acompound of Formula II or Formula IIa, or a salt thereof, and optionallycomprises a second, third, fourth, fifth or sixth therapeutic agent,wherein the optional first, second, third, fourth, fifth or sixththerapeutic agent is an antiretroviral agent selected from ofzidovudine, didanosine, lamivudine, zalcitabine, abacavir, stavudine,adefovir, adefovir dipivoxil, fozivudine, todoxil, emtricitabine,alovudine, amdoxovir, elvucitabine, nevirapine, delavirdine, efavirenz,loviride, immunocal, oltipraz, capravirine, lersivirine, GSK2248761,TMC-278, TMC-125, etravirine, saquinavir, ritonavir, indinavir,nelfinavir, amprenavir, fosamprenavir, brecanavir, darunavir,atazanavir, tipranavir, palinavir, lasinavir, enfuvirtide, T-20, T-1249,PRO-542, PRO-140, TNX-355, BMS-806, BMS-663068 and BMS-626529, 5-Helix,raltegravir, elvitegravir, GSK1349572, GSK1265744, vicriviroc (Sch-C),Sch-D, TAK779, maraviroc, TAK449, didanosine, tenofovir, lopinavir, ordarunavir.

As described herein, embodiments of the invention includecoadministering, whether simultaneously, sequentially or in combination,a first therapeutic agent that is a substituted benzoxaborole or saltthereof as described herein, preferably a substituted benzoxaborole ofFormula II or Formula IIa as described herein, or a pharmaceuticallyacceptable salt thereof, optionally in combination with a secondtherapeutic agent, optionally in combination with a third therapeuticagent, optionally in combination with a fourth therapeutic agent,optionally in combination with a fifth and/or a sixth therapeutic agent,to a subject exposed to or infected with a mycobacterium species,including a Mycobacterium tuberculosis species. In certain embodiments,the first therapeutic agent is a tricyclic benzoxaborole compound ofFormula II or Formula IIa as described herein or a pharmaceuticallyacceptable salt thereof, and the second and/or third and/or fourththerapeutic agent is an anti-tubercular agent. In certain embodiments,the mycobacterium species is a drug-resistant variant; in certainembodiments the mycobacterium species is a multi-drug resistant variant.

In other particular embodiments there is provided a method for killingmycobacteria comprising contacting the mycobacteria or an animal,including a human, exposed to or infected with a mycobacterium with afirst therapeutic agent that is a compound of Formula II or Formula IIaas described herein, or a pharmaceutically acceptable salt thereof,optionally contacting the cells or subject with a second therapeuticagent, optionally contacting the cells or subject with a thirdtherapeutic agent, optionally contacting the cells or subject with afourth therapeutic agent, optionally contacting the cells or subjectwith a fifth and/or a sixth therapeutic agent, such that contactingkills mycobacteria cells. In particular embodiments, the firsttherapeutic agent is a substituted benzoxaborole that is a compound ofFormula II or Formula IIa as described herein, or a pharmaceuticallyacceptable salt thereof and the optional second, third, fourth, fifthand/or sixth therapeutic agent is an anti-tubercular agent or a saltthereof. In other particular embodiments, the subject was exposed to oris infected with Mycobacterium tuberculosis.

Still other particular embodiments provide a method for inhibiting thereplication of mycobacterial cells, the method comprising contacting themycobacterial cells or an animal, including a human exposed to orinfected with a mycobacterial cells with a first therapeutic agent thatis a compound as described herein or a salt thereof, optionallycontacting the mycobacterial cells or animal with a second therapeuticagent, optionally contacting the mycobacterial cells or animal with athird therapeutic agent, optionally contacting the mycobacterial cellsor animal with a fourth therapeutic agent, optionally contacting themycobacterial cells or animal with a fifth and/or a sixth therapeuticagent, such that contacting inhibits the replication of themycobacterial cells. In particular embodiments, the first therapeuticagent is a substituted benzoxaborole that is a compound as describedherein or a salt thereof and the optional second, third, fourth, fifthand/or sixth therapeutic agent is an anti-tubercular agent or a saltthereof. In other particular embodiments, the subject was exposed to oris infected with Mycobacterium tuberculosis.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 is a world map indicating where, geographically, XDR-TB has beendocumented.

FIG. 2 shows transmission of tuberculosis.

FIG. 3 is a graph of MIC values (from Tables 1A and 1B) for Example 4G4-Cl against clinical isolates of M. tuberculosis.

FIG. 4 is a graph of MIC values (from Tables 2A, 2B, 2C and 2D) forExample 2 and Example 4 (G2-Br and G4-Cl, respectively) against clinicalisolates of M. tuberculosis.

Tables 1A and 1B provide MIC values for Example 4 G4-Cl tested against97 M. tuberculosis Clinical Isolates: Sensitive (A) and Resistant (B).Table 1A is MIC results for Example 4 against M. tuberculosis strainssensitive to known TB agents and Table 1B is MIC results for Example 4against M. tuberculosis strains resistant to one or more known TBagents. The resistance pattern of clinical isolates is indicated by thefollowing abbreviations H: Isoniazide, R: Rifampicin, T: Ethionamide, S:Streptomycin, E: Ethambutol, Z: Pyrazynamide, K: Kanamycin, A: Amikacinand CP: Capreomycin.

Tables 2A and 2B provide MIC values for Example 4 G4-Cl tested against40 strains of M. tuberculosis Clinical Isolates: Sensitive (A) andResistant (B). Table 2A is MIC results for Example 4 against M.tuberculosis strains sensitive to (Standard of Care TB agents?) andTable 2B is MIC results for Example 4 against M. tuberculosis strainsresistant to one or more known TB agents.

Tables 2C and 2D provide MIC values for Example 2 G2-Br tested against40 strains of M. tuberculosis Clinical Isolates: Sensitive (A) andResistant (B). Table 2C is MIC results for Example 2 against M.tuberculosis strains sensitive to known TB agent and Table 2D is MICresults for Example 2 against M. tuberculosis strains resistant to oneor more known TB agents. The resistance pattern of clinical isolates isindicated by the following abbreviations H: Isoniazide, R: Rifampicin,T: Ethionamide, S: Streptomycin, E: Ethambutol, Z: Pyrazynamide, K:Kanamycin, A: Amikacin and CP: Capreomycin.

Table 3 provides MIC values against non-Mycobacterial strains forCompounds of Formula II or Formula IIa.

Table 4A provides LeuRS inhibition IC50 values, MIC values against theM. tuberculosis standard strain Mtb H37Rv, toxicity values against humanHepG2 cells, and selectivity values for Certain Comparator TricyclicBenzoxaborole Compounds.

Table 4B provides the data classifications listed in Table 4A forCompounds of Formula II or Formula IIa.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

“Animal” as used herein means any of a kingdom (Animalia) of livingthings including many-celled organisms, including livestock and pets,including cattle, sheep, goats, dogs and cats, or a human, including animmune-suppressed human.

“Combination of the invention,” as used herein refers to thecombinations of compounds discussed herein, salts (e.g. pharmaceuticallyacceptable salts), prodrugs, solvates and hydrates of these compounds.

“Diastereomer” as used herein refers to one of a pair of stereoisomersthat is not mirror images of the other stereoisomer.

“Enantiomer” as used herein refers to one of a pair ofnon-superimposable racemic compounds (racemates) that is a mirror imageof the other enantiomer. Enantiomers have the property of rotating theplane of polarized light in one direction or another when in pure formbut as a racemic mixture, the mixture does not rotate the plane ofpolarized light.

“Effective” amount of a compound, combination thereof or formulationthereof, means an amount of a compound that is the active agent,including a combination of formulation thereof, such that the amount issufficient to provide the desired local or systemic effect. A“therapeutically effective” or “pharmaceutically effective” amountrefers to the amount of compound, including a combination or formulationthereof, sufficient to achieve a desired therapeutic or pharmaceuticalresult.

The term “pharmaceutically acceptable salt” is meant to include a saltof a compound described herein which is prepared with relativelynontoxic acids or bases, depending on the particular substituents foundon the compounds described herein. When compounds as described hereincontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino (suchas choline or diethylamine or amino acids such as d-arginine,l-arginine, d-lysine or l-lysine), or magnesium salt, or a similar salt.When compounds as described herein contain relatively basicfunctionalities, acid addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredacid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like (see, for example, Bergeet al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specific compounds as described herein containboth basic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompounds in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

In addition to salt forms, the invention provides compounds which are ina prodrug form. Prodrugs of the compounds described herein readilyundergo chemical changes under physiological conditions to provide thecompounds as described herein. Additionally, prodrugs can be convertedto the compounds of the invention by chemical or biochemical methods inan ex vivo environment.

Certain of the compounds of Formula 11 and Formula IIa may form acidaddition salts with one or more equivalents of the acid. The presentinvention includes within its scope all possible stoichiometric andnon-stoichiometric forms.

The compounds of Formula II and Formula IIa may be prepared incrystalline or non-crystalline form and, if crystalline, may optionallybe solvated, e.g. as the hydrate. This invention includes within itsscope stoichiometric solvates (e.g. hydrates) as well as compoundscontaining variable amounts of solvent (e.g. water).The subjectinvention also includes isotopically-labeled compounds which areidentical to those recited in Formula II and Formula IIa but for thefact that one or more atoms are replaced by an atom having an atomicmass or mass number different from the atomic mass or mass number mostcommonly found in nature. Examples of isotopes that can be incorporatedinto compounds as described herein include isotopes of hydrogen, carbon,nitrogen, oxygen, fluorine, iodine and chlorine such as ³H, ¹¹C, ¹⁴C,¹⁸F, ¹²³I or ¹²⁵I.

Compounds of the present invention and pharmaceutically acceptable saltsof said compounds that contain the aforementioned isotopes and/or otherisotopes of other atoms are within the scope of the present invention.Isotopically labeled compounds of the present invention, for examplethose into which radioactive isotopes such as ³H or ¹⁴C have beenincorporated, are useful in drug and/or substrate tissue distributionassays. Tritiated, ie. ³H, and carbon-14, ie. ¹⁴C, isotopes areparticularly preferred for their ease of preparation and detectability.¹¹C and ¹⁸F isotopes are particularly useful in PET (positron emissiontomography).

Because the compounds of Formula II and Formula IIa as described hereinare intended for use in pharmaceutical compositions it will readily beunderstood that they are each preferably provided in substantially pureform, for example at least 60% pure, more suitably at least 75% pure andpreferably at least 85%, especially at least 98% pure (% are on a weightfor weight basis). Impure preparations of the compounds may be used forpreparing the more pure forms used in the pharmaceutical compositions.

One embodiment provides a tricyclic benzoxaborole compound or a saltthereof having a structure according to Formula II:

wherein X is selected from chloro, fluoro, bromo and iodo; R¹ and R² areeach independently H, —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, and —CH(CH₃)₂.

One embodiment provides a compound of Formula II wherein X is chloro orbromo and R¹ and R² are independently selected from H, —CH₃, —CH₂CH₃,—CH₂CH₂CH₃, and —CH(CH₃)₂.

One embodiment provides a compound of Formula II or a salt thereof,wherein X is chloro or bromo; R¹ and R² are independently H, —CH₃, or—CH₂CH₃.

One embodiment provides a compound of Formula II or a salt thereof,wherein X is chloro or bromo; R¹ and R² are independently selected fromH and —CH₃.

One embodiment provides a compound of Formula II or a salt thereof,wherein X is fluoro or iodo; R¹ and R² are independently selected from Hand —CH₃.

Another embodiment provides a compound of Formula IIa

wherein X is fluoro, chloro, bromo or iodo, and R¹ and R² areindependently H or —CH₃, or a pharmaceutically acceptable salt thereof.

In one aspect the invention provides a pharmaceutical compositioncomprising a compound of Formula II, or a pharmaceutically acceptablesalt or solvate thereof, and one or more pharmaceutically acceptablecarriers, excipients or diluents.

Another aspect of the invention further provides a method of treatmentof a mycobacterial infection in a mammal, particularly in a human, whichmethod comprises administering to a mammal in need of such treatment aneffective amount of a first therapeutic agent that is a compound ofFormula II or a compound of Formula IIa, or a pharmaceuticallyacceptable salt or solvate thereof. Related embodiments further compriseadministering to a mammal in need of such treatment an effective amountof a first therapeutic agent that is a compound of Formula II or acompound of Formula IIa, or a pharmaceutically acceptable salt thereof,optionally administering in combination with an effective amount of asecond therapeutic agent, optionally administering in combination withan effective amount of a third therapeutic agent, optionallyadministering in combination with an effective amount of a fourththerapeutic agent, optionally administering in combination with aneffective amount of a fifth therapeutic agent, optionally administeringin combination with an effective amount of a sixth therapeutic agent.

In related aspects of the embodiment the optional second, third, fourth,fifth and sixth therapeutic agent is an anti-mycobacterial agent. Inrelated aspects, administering the first therapeutic agent andoptionally administering the second, third, fourth, fifth and sixththerapeutic agent occurs concurrently, or administering the firsttherapeutic agent and optionally administering the second, third,fourth, fifth and sixth therapeutic agent occurs sequentially. In otherrelated aspects of the invention, any one of the second, third, fourth,fifth or sixth therapeutic agent is selected from an antimicrobialagent, an antiviral agent, an anti-infective agent, an analgesic, avitamin, a nutritional supplement, an anti-inflammatory agent, ananalgesic, and an steroid.

The invention yet further provides a compound of Formula II, or apharmaceutically acceptable salt or solvate thereof, for use in thetreatment of a mycobacterial infection in a mammal, particularly in ahuman. In related aspects, the mammal is a human wherein themycobacterial infection is a Mycobacterium tuberculosis infection. Inother aspects, the human with a Mycobacterium tuberculosis infection isalso infected with a retrovirus, including a human immunodeficiencyvirus.

The invention still further provides the use of a compound of Formula IIor Formula IIa, or a pharmaceutically acceptable salt or solvatethereof, in the manufacture of a medicament for use in the treatment ofa mycobacterial infection in a mammal, particularly in a human.

The invention also provides a pharmaceutical composition comprising acompound of Formula II or Formula IIa, or a pharmaceutically acceptablesalt, or solvate thereof, and one or more pharmaceutically acceptablecarriers, excipients or diluents, for use in the treatment of amycobacterial infection in a mammal, particularly in a human.

The invention also provides a pharmaceutical composition comprising acompound of Formula II or Formula IIa, or a pharmaceutically acceptablesalt, or solvate thereof, and one or more pharmaceutically acceptablecarriers, excipients or diluents, for use in the treatment ofmycobacterial infections in a mammal, particularly in a human.

In another particular embodiment the substituted benzoxaborole in thecombination has a structure which is

or a pharmaceutically acceptable salt thereof.

In one particular embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In one particular embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In one particular embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In one particular embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

An embodiment of the invention provides a compound which is:

-   (3-bromo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;-   (S)-(3-bromo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;-   (3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;-   (S)-(3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;-   (3-chloro-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;-   (3-bromo-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;-   (3-bromo-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;-   (S)-(3-bromo-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;-   (3-chloro-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;-   (S)-(3-chloro-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;-   (3-fluoro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;-   (S)-(3-iodo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine;    or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the treatment of a mycobacterialinfection or condition occurs through inhibition of an editing domain ofan aminoacyl tRNA synthetase by means of binding to the editing activesite. In another exemplary embodiment, the treatment of a mycobacterialinfection or condition occurs through blocking of an editing domain ofan aminoacyl tRNA synthetase.

In a particular embodiment, the mycobacterial infection and/or diseaseis treated through oral administration of the combination of theinvention. In an exemplary embodiment, the mycobacterial infectionand/or disease is treated through intravenous administration of thecombination of the invention.

Pharmaceutical Formulations

In another aspect, the invention is a pharmaceutical formulation whichincludes: (a) a pharmaceutically acceptable excipient; (b) a combinationof the invention. In another aspect, the pharmaceutical formulationincludes: (a) a pharmaceutically acceptable excipient; and (b) acombination described herein. In another aspect, the pharmaceuticalformulation includes: (a) a pharmaceutically acceptable excipient; and(b) a combination described herein, or a salt, prodrug, hydrate orsolvate thereof. In another aspect, the pharmaceutical formulationincludes: (a) a pharmaceutically acceptable excipient; and (b) acombination described herein, or a salt, hydrate or solvate thereof. Inanother aspect, the pharmaceutical formulation includes: (a) apharmaceutically acceptable excipient; and (b) a combination describedherein, or a salt, hydrate or solvate thereof. In another aspect, thepharmaceutical formulation includes: (a) a pharmaceutically acceptableexcipient; and (b) a salt of a combination described herein. In anexemplary embodiment, the salt is a pharmaceutically acceptable salt. Inanother aspect, the pharmaceutical formulation includes: (a) apharmaceutically acceptable excipient; and (b) a prodrug of acombination described herein. In another aspect, the pharmaceuticalformulation includes: (a) a pharmaceutically acceptable excipient; and(b) a combination described herein. In an exemplary embodiment, thepharmaceutical formulation is a unit dosage form. In an exemplaryembodiment, the pharmaceutical formulation is a single unit dosage form.

In an exemplary embodiment, the pharmaceutical formulation is a unitdosage form. In an exemplary embodiment, the pharmaceutical formulationis a single unit dosage form. In an exemplary embodiment, thepharmaceutical formulation is a two unit dosage form. In an exemplaryembodiment, the pharmaceutical formulation is a three unit dosage form.In an exemplary embodiment, the pharmaceutical formulation is a fourunit dosage form. In an exemplary embodiment, the pharmaceuticalformulation is a five unit dosage form. In an exemplary embodiment, thepharmaceutical formulation is a six unit dosage form. In an exemplaryembodiment, the pharmaceutical formulation is a one, two, three, four,five, six or seven unit dosage form comprising a first unit dosage formand a second, third, fourth, fifth and/or sixth unit dosage form,wherein the first unit dosage form includes a) a therapeuticallyeffective amount of a compound as described herein and b) a firstpharmaceutically acceptable excipient; and the second, third, fourth,fifth, and/or sixth unit dosage form includes c) a therapeuticallyacceptable amount of an additional therapeutic agent that is ananti-mycobacterial agent and d) a second pharmaceutically acceptableexcipient.

Information regarding excipients of use in the formulations of theinvention can be found in Remington: The Science and Practice ofPharmacy, 21st Ed., Pharmaceutical Press (2011) which is incorporatedherein by reference.

Combinations

In an exemplary embodiment, the invention provides a) a firsttherapeutic agent that is a tricyclic benzoxaborole compound or saltthereof as described herein; b) a second therapeutic activity. Incertain embodiments, the second therapeutic agent is an antibacterialagent, more specifically an anti-tubercular agent, more specifically ananti-M. tuberculosis agent.

In an exemplary embodiment, the combination is part of a pharmaceuticalformulation described herein. Such conditions are known to one skilledin the art and specific conditions are set forth in the Examplesappended hereto.

Dosage Forms of the Combination

The individual components of the combinations of the invention, forexample, a combination described herein, may be administered eithersimultaneously or sequentially in a unit dosage form. The unit dosageform may be a single or multiple unit dosage form. In an exemplaryembodiment, the invention provides a combination in a single unit dosageform. An example of a single unit dosage form is a capsule wherein boththe tricyclic benzoxaborole compound and additional therapeutic agentare contained within the same capsule. In an exemplary embodiment, theinvention provides a combination in a two unit dosage form. An exampleof a two unit dosage form is a first capsule which contains thetricyclic benzoxaborole compound and a second capsule which contains theadditional therapeutic agent. Thus the term ‘single unit’ or ‘two unit’or ‘multiple unit’ refers to the object which the patient ingests, notto the interior components of the object. Appropriate doses of tricyclicbenzoxaborole compound will be readily appreciated by those skilled inthe art. Appropriate doses of an additional therapeutic agent that isnot a compound of Formula II or Formula IIA will be readily appreciatedby those skilled in the art. In one particular embodiment, the tricyclicbenzoxaborole compound is present in the combination in atherapeutically effective amount. In one particular embodiment, theadditional therapeutic agent that is not a compound of Formula II orFormula IIA is present in the combination in an amount sufficient tokill or reduce the presence, amount or growth rate of mycobacteriaexposed to the substituted benzoxaborole, including M. tuberculosis.

Additional Therapeutic Agent(s) in the Combination

The combinations of the invention, for example, a combination describedherein, may also include an additional therapeutic agent or therapeuticagents. The invention thus provides, in a further aspect, a combinationcomprising a tricyclic benzoxaborole compound described herein or apharmaceutically acceptable salt thereof, and at least one additionaltherapeutic agent. The invention thus provides, in a further aspect, acombination comprising a tricyclic benzoxaborole compound describedherein or a pharmaceutically acceptable salt thereof, and at least oneadditional therapeutic agent. In an exemplary embodiment, the additionaltherapeutic agent is an antimycobacterial agent. In one aspect, theinvention comprises: a) a combination of the invention; and b) at leastone additional therapeutic agent. In another exemplary embodiment, theinvention comprises: a) a combination of the invention; b) a firstadditional therapeutic agent; and c) a second additional therapeuticagent. In another exemplary embodiment, the invention comprises: a) acombination of the invention; b) a first additional therapeutic agent;c) a second additional therapeutic agent; and d) a third additionaltherapeutic agent. The first additional therapeutic agent or secondadditional therapeutic agent or third additional therapeutic agent maybe selected from the additional therapeutic agents described herein.

The combinations may conveniently be presented for use in the form of apharmaceutical formulation. In a further aspect of the present inventionthere is provided a pharmaceutical combination comprising a compound ofFormula II, or a pharmaceutically acceptable salt or solvate thereof,together with one or more additional therapeutic agents, and one or morepharmaceutically acceptable carriers, excipients or diluents. Theindividual components of such combinations may be administered eithersequentially or simultaneously in separate or combined pharmaceuticalFormulations by any convenient route.

When an additional therapeutic agent is used with a combination asdescribed herein against the same disease state, the dose of eachcompound may differ from that when the compound is used alone.Appropriate doses will be readily appreciated by those skilled in theart. It will be appreciated that the amount of a compound as describedherein required for use in treatment will vary with the nature of thecondition being treated and the age and the condition of the patient andwill be ultimately at the discretion of the attendant physician orveterinarian.

Preparation of Boron-Containing Compounds

Compounds of use in the invention can be prepared using commerciallyavailable starting materials, known intermediates, or by using thesynthetic methods described herein, or published in references describedand incorporated by reference herein, such as U.S. Pat. No. 7,816,344and PCT Pat. Pubs. WO2010080558 and WO2011127143. The general proceduresused to synthesize the compounds of Formula II and Formula IIa, aredescribed in reaction Schemes 1-5 and are illustrated in the Examples.

Certain tricyclic benzoxaborole compounds may be prepared by a Mitsunobureaction to convert the hydroxyl substituent of2-bromo-3-hydroxy-benzaldehyde into the tetrahydropyranyloxyethoxy etherwith tetrahydropyranyloxyethanol in triphenylphosphine (PPh₃),tetrahydrofuran (THF) and diisopropyl azodicarboxylate (DIAD), followedby Miyaura borylation of the bromine substituent usingbis(pinocolato)diboron diboron (B₂pin₂) with a palladium catalyst(PdCl₂) and potassium acetate (KOAc), and then reductive ring closure toform the tricyclic compound using sodium borohydride (NaBH₄) inanhydrous methanol (MeOH), as outlined in Scheme 1.

THP-protected alkyl bromide may also be used to attach a substituent tohydroxybenzaldehyde via a SN2 reaction to prepare tricyclicbenzoxaborole compounds. Examples of the use of a SN2 reaction forpreparing tricyclic benzoxaborole compounds can be seen in the Examplesdescribed below, such as in Example 1, step (b) and Example 4, Method B,step (c).

Other tricyclic benzoxaborole compounds as described herein may beprepared as outlined in Schemes 2 and 3, wherein a nitro-aldol reactionis performed on the aldehyde substituent of, for example,3-(2-benzyloxy-ethoxy)-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzaldehydeusing nitromethane (MeNO₂) with base (NaOH) to prepare thenitro-substituted benzyl-protected benzoxaborole compound, followed byring-closure to and reduction of the nitro substituent to the amine withPd(OH)₂/C in glacial acetic acid to form the desired tricyclicbenzoxaborole compound.

Other tricyclic benzoxaborole compounds as described herein may beprepared as outlined in Scheme 4.

Other tricyclic benzoxaborole compounds as described herein may beprepared as outlined in Scheme 5.

Alternatively, certain tricyclic benzoxaborole compounds may be preparedas outlined in Scheme 6. A mixture of (S)-tert-butyl((7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(13.25 kg) and NCS (8.75 kg) in dichloroethane (132.5 L) was heated at70° C. until the reaction judged complete by HPLC. The mixture wasconcentrated under reduced pressure, cooled to 25° C. and acetone (106L) added. The slurry was filtered, washing with acetone (26.5 L). Thewet cake was slurried in water (13.25 L) and 1,4-dioxane (66.25 L),heated to 50° C. for 20-30 minutes, cooled to 15° C., filtered and thecake washed with 1,4-dioxane (26.5 L). The wet cake was dissolved inmethanol (68.9 L), filtered and the filtrate concentrated under reducedpressure. Methyl tertiary butyl ether (66.25 L) was added to the residueand the mixture concentrated under reduced pressure. Methyl tertiarybutyl ether (78.7 L), isopropanol (8.7 L) and sulphuric acid (4.6 L)were added, the mixture heated to 50° C. and stirred until the sulphatecontent was 24.32-29.72%. The mixture was cooled to 25° C., stirred for1 hour, filtered, the cake washed with methyl tertiary butyl ether (17.5L) and dried to give the desired product (42%).

As can be seen in Scheme 6, in this route the final steps 10/11 and12/13 are replaced with alternative final steps, eliminating theprotection/deprotection steps.

Composition and Formulations

The compounds as described herein may be formulated for administrationin any convenient way for use in human or veterinary medicine, byanalogy with formulation of anti-mycobacterial agents, or formulation ofother anti-tubercular agents.

The compounds described herein will normally, but not necessarily, beformulated into pharmaceutical compositions prior to administration to apatient. In one aspect, the invention is directed to a pharmaceuticalcomposition comprising a compound of Formula II or compound of FormulaIIa, or a pharmaceutically acceptable salt. In another aspect theinvention is directed to a pharmaceutical composition comprising acompound of Formula II or a compound of Formula IIa, or apharmaceutically acceptable salt, and one or more pharmaceuticallyacceptable carriers, excipients or diluents. The carrier, excipient ordiluent must be “acceptable” in the sense of being compatible with theother ingredients of the Formulation and not deleterious to therecipient thereof.

The pharmaceutical compositions described herein include those in a formadapted for oral, or parenteral use and may be used for the treatment ofa mycobacterial infection in a mammal including a human.

The pharmaceutical compositions described herein include those in a formadapted for oral, topical or parenteral use and may be used for thetreatment of mycobacterial infections in a mammal including a human.

The composition may be formulated for administration by any convenientroute. For the treatment of tuberculosis, the compositions may be in theform of tablets, capsules, powders, granules, lozenges, aerosols orliquid preparations, such as oral or sterile parenteral solutions orsuspensions.

Tablets and capsules for oral administration may be in unit dosepresentation form, and may contain conventional excipients such asbinding agents, for example syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinylpyrrolidone; fillers, for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricants, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants, for example potato starch; or acceptable wettingagents such as sodium lauryl sulphate. The tablets may be coatedaccording to methods well known in normal pharmaceutical practice. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives, such as suspending agents, for example sorbitol,methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose,carboxymethyl cellulose, aluminium stearate gel or hydrogenated ediblefats, emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, oily esters such as glycerine, propylene glycol, orethyl alcohol; preservatives, for example methyl or propylp-hydroxybenzoate or sorbic acid, and, if desired, conventionalflavouring or colouring agents.

Suppositories will contain conventional suppository bases, e.g.cocoa-butter or other glyceride.

For parenteral administration, fluid unit dosage forms are preparedutilizing the compound and a sterile vehicle, water being preferred. Thecompound, depending on the vehicle and concentration used, can be eithersuspended or dissolved in the vehicle. In preparing solutions thecompound can be dissolved in water for injection and filter sterilisedbefore filling into a suitable vial or ampoule and sealing.

In one aspect of the invention, agents such as a local anaesthetic,preservative and buffering agents can be dissolved in the vehicle. Toenhance the stability, the composition can be frozen after filling intothe vial and the water removed under vacuum. The dry lyophilized powderis then sealed in the vial and an accompanying vial of water forinjection may be supplied to reconstitute the liquid prior to use.Parenteral suspensions are prepared in substantially the same mannerexcept that the compound is suspended in the vehicle instead of beingdissolved and sterilization cannot be accomplished by filtration. Thecompound can be sterilised by exposure to ethylene oxide beforesuspending in the sterile vehicle. Advantageously, a surfactant orwetting agent is included in the composition to facilitate uniformdistribution of the compound.

The compositions may contain from 0.1% by weight, preferably from 10-60%by weight, of the active material, depending on the method ofadministration. Where the compositions comprise dosage units, each unitwill preferably contain from 20-1000 mg of the active ingredient. Thedosage as employed for adult human treatment will typically range from50 to 300 mg per day, for instance 150 to 200 mg per day depending onthe route and frequency of administration. Such a dosage corresponds to0.5 to 5 mg/kg per day. Preferably the dosage is from 0.5 to 2 mg/kg perday and more preferably the dose is less than 1 mg/kg per day.

The compound of Formula II or Formula IIa, or a pharmaceuticallyacceptable pharmaceutically acceptable salt or solvate thereof, may bethe sole therapeutic agent in the compositions described herein, or itmay be present in the Formulation in combination with one or moreadditional therapeutic agents. The invention thus provides, in a furtheraspect, a combination comprising a compound of Formula II, or apharmaceutically acceptable salt, solvate thereof together with one ormore additional therapeutic agents.

The one or more additional therapeutic agent is, for example, an agentuseful for the treatment of tuberculosis in a mammal. Examples of suchtherapeutic agents include, rifampin, pyrazinamide, ethambutol,moxifloxacin, rifapentine, clofazimine, bedaquiline (TMC207),nitroimidazo-oxazine PA-824, delamanid (OPC-67683), oxazolidinone suchas linezolid, tedizolid, radezolid, sutezolid (PNU-100480), andposizolid (AZD-5847), EMB analogue SQ109, a benzothiazinone, adinitrobenzamide and an antiviral agent including an antiretroviralagent, or any TB agent being developed for the treatment of TB with apositive response in Phase IIa EBA trials, or any TB agent underdevelopment by the Global Alliance for Tuberculosis.

When a compound of Formula II or Formula IIa, or a pharmaceuticallyacceptable salt or solvate thereof is used in combination with one ormore additional therapeutic agents, the dose of the compound or agentmay differ from that when the compound or agent is used alone.Appropriate doses will be readily appreciated by those skilled in theart. It will be appreciated that the amount of a compound describedherein and the one or more additional therapeutic agents required foruse in treatment will vary with the nature of the condition beingtreated and the age and the condition of the patient and will beultimately at the discretion of the attendant physician or veterinarian.

The combinations may conveniently be presented for use in the form of apharmaceutical Formulation. In a further aspect of the present inventionthere is provided a pharmaceutical combination comprising a compound ofFormula II, or a pharmaceutically acceptable salt or solvate thereof,together with one or more additional therapeutic agents, and one or morepharmaceutically acceptable carriers, excipients or diluents. Theindividual components of such combinations may be administered eithersequentially or simultaneously in separate or combined pharmaceuticalFormulations by any convenient route.

When administration is sequential, either the compound of the presentinvention or one or more additional therapeutic agent may beadministered first. When administration is simultaneous, the combinationmay be administered either in the same or different pharmaceuticalcomposition. When combined in the same Formulation it will beappreciated that the compound and agents must be stable and compatiblewith each other and the other components of the Formulation. Whenformulated separately they may be provided in any convenientformulation, conveniently in such manner as are known for such compoundsin the art.

Methods of Inhibiting Bacterial Growth or Killing Bacteria

The combinations of the invention are expected to exhibit potencyagainst mycobacteria and therefore have the potential to killmycobacteria and/or inhibit the replication of mycobacteria. Thecombinations of the invention are expected to exhibit potency againstmycobacteria possessing resistance to standard-of-careanti-mycobacterial agents, and thus have the potential to killmycobacteria and/or inhibit the replication of such “resistant”mycobacteria. In aspects of the invention, compounds as described hereinpossess a remarkable activity against a selection of drug sensitivemycobacterial isolates, including, MDR-TB (multidrug resistant TB) andXDR-TB (extensively-drug resistant TB) clinical isolates, exhibiting MICvalues of <0.32 μM and the majority have MIC values at between 0.04-0.08μM in 96 isolates investigated.

In a further aspect, the invention provides a method of killingmycobacteria and/or inhibiting replication of mycobactera or a method oftreating a mycobacterial infection in an animal such as livestock andpets, including cattle sheep, goats, dogs and cats, or a human,including an immune-suppressed human said method comprising: contactingthe mycobactera with an effective amount of a combination as describedherein, thereby killing the mycobacteria and/or inhibiting replicationof the mycobacteria, or said method comprising administering to theanimal with the mycobacterial infection a therapeutically effectiveamount of a combination of the invention, wherein the combinationcomprises a compound of Formula II or a compound of Formula IIa, or apharmaceutically acceptable salt thereof. In an exemplary embodiment,the combination is part of a pharmaceutical formulation describedherein. In another exemplary embodiment, the contacting occurs underconditions which permit entry of the combination into the mycobacterium.

In an exemplary embodiment, the mycobacteria is killed or itsreplication is inhibited, or the mycobacterial infection is treated,through oral administration of a combination as described herein. In anexemplary embodiment, the mycobacteria is killed or its replication isinhibited, or the mycobacterial infection is treated, throughintravenous administration of a combination as described herein. In anexemplary embodiment, the mycobacterium is killed or its replication isinhibited, or the mycobacterial infection is treated, throughsubcutaneous administration of a combination as described herein,wherein the combination comprises a compound of Formula II or a compoundof Formula IIa, or a pharmaceutically acceptable salt thereof.

In exemplary embodiments, the mycobacteria is contacted or themycobacterial infection is treated with a combination as describedherein comprising a first therapeutic agent that is a compound ofFormula II or a compound of Formula IIa, or salt thereof, and optionallycomprising a second, third, fourth, fifth and sixth therapeutic agent ina population of mycobacteria comprising a resistant mycobacterium with amutation conferring resistance to any one or more of the optionalsecond, third, fourth, fifth and sixth therapeutic agent. In relatedembodiments, the optional second, third, fourth, fifth and sixththerapeutic agent, or a salt thereof, is an anti-mycobacterial agent,particularly a known anti-mycobacterial agent, more preferably astandard-of-care anti-mycobacterial agent.

In another exemplary embodiment, there is provided a method of killingand/or inhibiting replication of mycobacteria that causes or isassociated with a disease in an animal, or a method of treating amycobacterial infection in an animal, the method comprising contactingthe mycobacteria with an effective amount of a compound of Formula II orFormula IIa or a salt thereof, so as to kill and/or prevent replicationof the mycobacterium, or administering to the animal a therapeuticallyeffective amount of a compound of Formula II or Formula IIa or a saltthereof, wherein the mycobacteria is selected from Mycobacteriumtuberculosis, Mycobacterium avium including subspecies (subsp.)Mycobacterium avium subsp. avium, Mycobacterium avium subsp.hominissuis, Mycobacterium avium subsp. silvaticum, and Mycobacteriumavium subsp. paratuberculosis; Mycobacterium balnei, Mycobacteriumsherrisii, Mycobacterium africanum, Mycobacterium microti, Mycobacteriumsilvaticum, Mycobacterium colombiense, Mycobacterium indicus pranii,Mycobacterium gastri, Mycobacterium gordonae, Mycobacterium hiberniae,Mycobacterium nonchromagenicum, Mycobacterium terrae, Mycobacteriumtrivial, Mycobacterium kansasii; Mycobacterium malmoense; Mycobacteriumsimiae; Mycobacterium triplex, Mycobacterium genavense, Mycobacteriumflorentinum, Mycobacterium lentiflavum, Mycobacterium palustre,Mycobacterium kubicae, Mycobacterium parascrofulaceum, Mycobacteriumheidelbergense, Mycobacterium interjectum, Mycobacterium szulgai;Mycobacterium branderi, Mycobacterium cookie, Mycobacterium celatum,Mycobacterium bohemicum, Mycobacterium haemophilum, Mycobacteriumlepraemurium, Mycobacterium lepromatosis, Mycobacterium botniense,Mycobacterium chimaera, Mycobacterium conspicuum, Mycobacterium doricum,Mycobacterium forcinogenes, Mycobacterium heckeshornense, Mycobacteriumlacus, Mycobacterium monacense, Mycobacterium montefiorense,Mycobacterium murale, Mycobacterium nebraskense, Mycobacteriumsaskatchewanenese, Mycobacterium scrofulaceum, Mycobacterium shimoidel,Mycobacterium tusciae, Mycobacterium xenopi, Mycobacterium intermedium,Mycobacterium bolletii, Mycobacterium fortuitum, Mycobacterium foruitumsubsp. acetamidolyticum, Mycobacterium boenickei, Mycobacteriumperigrinum, Mycobacterium porcinum, Mycobacterium senegalense,Mycobacterium septicum, Mycobacterium neworleansense, Mycobacteriumhoustonense, Mycobacterium mucogenicum, Mycobacterium mageritense,Mycobacterium brisbanense, Mycobacterium cosmeticum, Mycobacteriumparafortuitum, Mycobacterium austroafricanum, Mycobacterium diemhoferi,Mycobacterium hodieri, Mycobacterium neoaurum, Mycobacteriumprederkisbergense, Mycobacterium aurum, Mycobacterium vaccae,Mycobacterium chitae, Mycobacterium fallax, Mycobacterium confluentis,Mycobacterium flavenscens, Mycobacterium madagascariense, Mycobacteriumphlei, Mycobacterium smegmatis, Mycobacterium goodie, Mycobacteriumcolinskui, Mycobacterium thermoresistbile, Mycobacterium gadium,Mycobacterium kormossense, Mycobacterium obuense, Mycobacterium sphagni,Mycobacterium agri, Mycobacterium aichiense, Mycobacterium alvei,Mycobacterium arupense, Mycobacterium brumae, Mycobacteriumcanariasense, Mycobacterium chubuense, Mycobacterium conceptionense,Mycobacterium duvalii, Mycobacterium elephantis, Mycobacterium gilvum,Mycobacterium hassiacum, Mycobacterium holsaticum, Mycobacteriumimmunogenum, Mycobacterium massiliense, Mycobacterium moriokaense,Mycobacterium psychrotoleranse, Mycobacterium pyrenivorans,Mycobacterium vanbaalenii, Mycobacterium pulveris, Mycobacteriumarosiense, Mycobacterium aubagnense, Mycobacterium caprae, Mycobacteriumchlorophenolicum, Mycobacterium fluoroanthenivorans, Mycobacteriumkumamotonense, Mycobacterium novocastrense, Mycobacterium parmense,Mycobacterium phocaicum, Mycobacterium poriferae, Mycobacteriumrhodesiae, Mycobacterium seolense, Mycobacterium tokalense,Mycobacterium xenopi; Mycobacterium scrofulaceum; Mycobacteriumabscessus; Mycobacterium chelonae; Mycobacterium haemophilum;Mycobacterium leprae; Mycobacterium marinum; Mycobacterium fortuitum;Mycobacterium bovis; Mycobacterium ulcerans; Mycobacteriumpseudoshottsii, Mycobacterium shottsii, Mycobacterium intracellulare;Mycobacterium tuberculosis complex (MTC); Mycobacteriumavian-intracellulare complex (MAIC) member and Mycobacterium aviumcomplex (MAC) member.

In related aspects, the mycobacterium is Mycobacterium tuberculosis. Inother aspects, the mycobacterium is Mycobacterium avium, Mycobacteriumkansasii, Mycobacterium malmoense, Mycobacterium simiae, Mycobacteriumszulgai, Mycobacterium xenopi, Mycobacterium scrofulaceum, Mycobacteriumabscessus, Mycobacterium chelonae, Mycobacterium haemophilum,Mycobacterium leprae, Mycobacterium marinum, M. fortuitum, Mycobacteriumbovis, M. bovis BCG, M. africanum, M. canetti, M. caprae, M. microti, M.pinnipedi, M. leprae or Mycobacterium ulcerans. In related embodiments,the mycobacterium is a subspecies (subsp.) of Mycobacterium avium,including Mycobacterium avium subsp. avium, Mycobacterium avium subsp.hominissuis, Mycobacterium avium subsp. silvaticum, and Mycobacteriumavium subsp. paratuberculosis. In another related embodiment, themycobacterium is Mycobacterium intracellulare. In further relatedembodiments, the mycobacterium is a member of the Mycobacteriumtuberculosis complex. (MTC) the Mycobacterium avium complex (MAC) or theMycobacterium avian-intracellulare complex (MAIC). In relatedembodiments, the mycobacterium is a non-tuberculosis complex or clade,including: Mycobacterium avium complex; Mycobacterium gordonae clade;Mycobacterium kansasii clade; Mycobacterium chelonae clade;Mycobacterium fortuitum clade; Mycobacterium parafortuitum clade; andMycobacterium vaccae clade.

In an exemplary embodiment, the mycobacteria in the methods describedherein comprises a resistant mycobacterium. In an exemplary embodiment,the resistant mycobacterium is a mutation of a mycobacteria describedherein.

Methods of Treating and/or Preventing Disease

The combinations of the present invention exhibit potency againstmycobacteria, and therefore have the potential to achieve therapeuticefficacy in animals, including humans.

In another aspect, the invention provides a method of treating and/orpreventing a disease. The method includes administering to the animal atherapeutically effective amount of a combination of the invention,sufficient to treat and/or prevent the disease. In an exemplaryembodiment, the combination of the invention can be used in human orveterinary medical therapy, particularly in the treatment or prophylaxisof mycobacterial-associated disease. In an exemplary embodiment, thecombination is described herein.

In another exemplary embodiment, the animal is as defined herein. Inanother exemplary embodiment, the disease a systemic disease or acutaneous disease. In another exemplary embodiment, the disease is arespiratory disease.

Abbreviations

In describing the invention, chemical elements are identified inaccordance with the Periodic Table of the Elements. Abbreviations andsymbols utilized herein are in accordance with the common usage of suchabbreviations and symbols by those skilled in the chemical arts. Thefollowing abbreviations are used herein:

-   -   AcOH acetic acid    -   Ac₂O acetic anhydride    -   AIBN 2-2′-Azoisobutyronitrile    -   BOC N-tert-butoxycarbonyl    -   BOC anhydride di-tert-butyl dicarbonate    -   B₂pin₂ bis(pinacolato)diboron diboron, also known as        4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane    -   Celite® a filter aid composed of acid-washed diatomaceous        silica, (a trademark of Manville Corp., Denver, Colo.)    -   CTAB cetyltrimethylammonium bromide    -   DCM dichloromethane    -   DIAD diisopropyl azodicarboxylate    -   DIBAL-H diisobutyl aluminium hydride    -   DME dimethoxyethane    -   DCE dichloroethane    -   DMF dimethylformamide    -   DMSO-d6 deuterated dimethylsulfoxide    -   DMSO dimethylsulfoxide    -   ESI Electrospray ionization    -   ES MS Electrospray mass spectrometry    -   Et₂O diethyl ether    -   EtOH ethanol    -   EtOAc, EA ethyl acetate    -   h hours    -   HPLC high performance liquid chromatography    -   KOAc potassium acetate    -   LCMS Liquid chromatography mass spectroscopy    -   mCPBA meta-chloro perbenzoic acid    -   MeNO₂ nitromethane    -   MeOH methanol    -   NBS N-bromosuccinimide    -   NCS N-chlorosuccinimide    -   NIS N-iodosuccinimide    -   NXS N-halosuccinimide    -   NaBH(OAc)₃ sodium triacetoxyborohydride    -   NMR Nuclear Magnetic Resonance spectroscopy    -   PE petroleum ether    -   PPh₃ triphenylphosphine    -   rt or r.t. room temperature    -   RT retention time    -   SFC supercritical fluid chromatography    -   t-BuOMe methyl t-butyl ether    -   TFA trifluoroacetic acid    -   THF tetrahydrofuran    -   uv ultraviolet

EXAMPLES

The following examples illustrate the invention. These Examples are notintended to limit the scope of the invention, but rather to provideguidance to the skilled artisan to prepare and use the compounds,compositions, and methods of the invention. While particular embodimentsof the invention are described, the skilled artisan will appreciate thatvarious changes and modifications can be made. References topreparations carried out in a similar manner to, or by the generalmethod of, other preparations, may encompass variations in routineparameters such as time, temperature, workup conditions, minor changesin reagent amounts etc.

Proton nuclear magnetic resonance (¹H NMR) spectra were recorded, andchemical shifts are reported in parts per million (δ) downfield from theinternal standard tetramethylsilane (TMS). Abbreviations for NMR dataare as follows: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet,dd=doublet of doublets, dt=doublet of triplets, app=apparent, br=broad.Mass spectra were obtained using electrospray (ES) ionizationtechniques. All temperatures are reported in degrees centigrade.

Reactions involving metal hydrides including lithium hydride, lithiumaluminium hydride, di-isobutylaluminium hydride, sodium hydride, sodiumborohydride and sodium triacetoxyborohydride are carried out under argonunless otherwise specified.

Synthesis Example 13-bromo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride (G1-Br)

(a) 2-bromo-3-hydroxybenzaldehyde

A suspension of 3-hydroxybenzaldehyde (5 g, 40 mmol), iron powder (172mg, 3 mmol) and sodium acetate (6.72 g, 80 mmol) in acetic acid (40 mL)was warmed until a clear solution was obtained and then cooled to roomtemperature. To this mixture was added dropwise a solution of bromine(7.2 g, 45 mmol) in glacial acetic acid (10 mL) over 15 min. After theaddition, the reaction mixture was stirred for 2 h and then poured intoice-water. The resulting mixture was extracted with dichloromethane(3×50 mL). The combined extracts were dried over anhydrous Na₂SO₄ andconcentrated. The residue was re-crystallized from dichloromethane toafford the product (2.3 g, 28%). ¹H NMR (300 MHz, DMSO-d₆): δ 10.75 (s,1H), 10.26 (s, 1H), 7.38-7.24 (m, 3H).

(b) 2-bromo-3-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)benzaldehyde

Dihydropyran (1.26 g, 15 mmol) was added dropwise at 0° C. to2-bromoethanol (1.875 g, 15 mmol). The mixture was stirred 30 min at 0°C. and then 2 h at rt. 2-bromo-3-hydroxy benzaldehyde (2 g, 10 mmol) wasadded to this mixture, followed by potassium carbonate (1.518 g, 11mmol), potassium iodide (332 mg, 2 mmol) and dry DMF (20 mL). Thereaction was stirred at 70° C. overnight. The solution was cooled to rtand diluted with diethyl ether (100 mL). The inorganic salts wereremoved by filtration and the filtrate was diluted with hexanes (100mL). The organic layer was washed with water (50 mL×3), and thenconcentrated to dryness under reduced pressure. The residue was purifiedby column chromatography on silica gel using ethyl acetate and petroleumether as eluents to give the target compound (3 g, 92%) as a yellow oil.MS (ESI) m/z=351 [M+23]⁺, Rf=0.7 (PE:EA=3). ¹H NMR (300 MHz, DMSO-d₆): δ10.29 (s, 1H), 7.50-7.41 (m, 3H), 4.75 (s, 1H), 4.31-4.28 (m, 2H),4.00-3.94 (m, 1H), 3.82-3.75 (m, 2H), 3.47-3.43 (m, 1H), 1.73-1.50 (m,6H).

(c)3-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde

A solution of2-bromo-3-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)benzaldehyde (160 g,0.49 mol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane)(249 g, 0.98 mol), Pd(dppf)Cl₂ (20 g, 24.5 mmol) and KOAc (144 g, 1.47mol) in DMF (2.0 L) was stirred at 90° C. overnight. Then the reactionmixture was treated with water (4 L) and then extracted with EtOAc(3×1.5 L). The combined organic layers were washed with brine (250 mL),dried over anhydrous Na₂SO₄ and concentrated to dryness in vacuo. Theresidue was purified by column chromatography on silica gel (petroleumether:ethyl acetate=10:1 to 2:1) to give the target compound as a yellowoil (88 g, yield 48%). MS (ESI) m/z=317 [M+H]⁺, Rf=0.4 (PE:EA=3). ¹H NMR(300 MHz, DMSO-d₆): δ 9.88 (s, 1H), 7.60-7.51 (m, 2H), 7.31-7.28 (d,1H), 4.64-4.63 (m, 1H), 4.16-4.13 (m, 2H), 4.00-3.94 (m, 1H), 3.82-3.75(m, 2H), 3.47-3.43 (m, 1H), 1.73-1.50 (m, 6H), 1.29 (m, 12H).

(d) 2-(nitromethyl)-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulene

To a solution of NaOH (4.8 g, 0.12 mol) in water (100 mL) was addednitromethane (18.3 g, 0.3 mol) at 5-10° C. After stirring for 15 min at5-10° C., cetyltrimethylammonium bromide (CTAB) (2.2 g, 6 mmol) wasadded to the reaction mixture and followed by the addition of3-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde(45 g, 0.12 mol) at 5-10° C. The reaction mixture was stirred at rt for5 h. The reaction mixture was acidified to pH=1 using dilutedhydrochloric acid and stirred at rt overnight. The reaction mixture wasfiltered to give the target compound (14.5 g, 51%) as a white solid. ¹HNMR (300 MHz, DMSO-d₆): δ 7.50-7.45 (t, 1H), 7.16-7.13 (d, 1H),6.91-6.88 (d, 1H), 5.91-5.88 (m, 1H), 5.37-5.31 (m, 1H), 4.69-4.61 (m,2H), 4.41-4.14 (m, 3H).

(e) (7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride

A solution of2-(nitromethyl)-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulene (1.5g, 6.4 mmol), Raney Ni (200 mg) and 2 M NH₃ in EtOH (5 mL) in ethanol(40 mL) was shaken under an atmosphere of H₂ for 2 h at rt. The mixturewas filtered through a bed of Celite and the filtrate was concentratedin vacuo. The crude amine was dissolved in EtOH (20 mL) and a saturatedsolution of HCl (gas) in Et₂O (30 mL) was added immediately. After 1 h,the suspension was filtered and the resulting solid was washed withacetonitrile/hexanes (2:1, 2×20 mL) to give the compound as a whitesolid (700 mg, 45%). MS (ESI) m/z=206/224 [M+H]⁺.

(f) tert-butyl((7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate

To a mixture of(7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride (700 mg, 2.9 mmol) and triethylamine (878.7 mg, 8.7 mmol)in dichloromethane (10 mL) at 0° C. was added di-tert-butyl dicarbonate(948 mg, 4.35 mmol) and the mixture was stirred for 2 h at roomtemperature. The reaction was quenched with sat. NaHCO₃ (15 mL) and theresulting mixture was extracted with EtOAc (3×20 mL). The combinedorganic layers were dried over anhydrous Na₂SO₄ and concentrated invacuo. The residue was purified by flash-column chromatography usingethyl acetate and petroleum ether as eluents to give the desired product(500 mg, 56%). MS (ESI) m/z=250 [M−56]⁺.

(g) tert-butyl((3-bromo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate

To a solution of tert-butyl((7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(0.5 mg, 1.64 mmol) and NBS (354 mg, 2.0 mmol) in acetonitrile (15 mL)was added AlBN (27 mg) and the mixture was stirred for 1 h at 90° C. Thereaction mixture was then concentrated under vacuum and the residue waspurified by preparatory-HPLC to give the desired product (300 mg, 50%).MS (ESI) m/z=328/330 [M−56]⁺.

(h) Title Compound

A mixture of tert-butyl((3-bromo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(0.2 g, 0.522 mmol) in saturated HCl (gas) in Et₂O (10 mL) was stirredat rt for 1 h and concentrated to dryness (water bath temperature<30°C.). The residue was triturated with acetonitrile (2×5 mL) and the whitesolid was dried under high vacuum to give the product (140 mg, 83%) as awhite solid. ¹H NMR (300 MHz, DMSO-d₆): δ 8.36 (s, 3H), 7.64-7.61 (d,1H), 6.93-6.90 (d, 1H), 5.51-5.49 (d, 1H), 4.69 (m, 1H), 4.36-4.23 (m,3H), 3.62 (m, 1H), 3.05-3.01 (m, 1H). MS (ESI) m/z=284/286 [M+H]⁺.

Example 2(S)-(3-bromo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride (G2-Br)

Method A (a) Title Compound

The racemic compoundtert-butyl((3-bromo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(Example 1, (g)) was separated via supercritical fluid chromatography(SFC) (chiral column CHIRALCEL OJ-H, eluted with MeOH (15%) and CO₂(85%) and two chiral compounds (S)-tert-butyl((3-bromo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(second eluting isomer, RT=3.8 min) and (R)-isomer tert-butyl((3-bromo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(first eluting isomer, RT=3.3 min) were obtained. Each of the chiralcompounds (1.2 g, 3.13 mmol) in saturated HCl (gas) in Et₂O (20 mL) wasstirred at room temperature for 1 h and concentrated to dryness (waterbath <30° C.). The residue was washed with acetonitrile (2×5 mL) and thewhite solid was dried under high vacuum to give the product (900 mg,90%) as a white solid. MS (ESI) m/z=284/286 [M+H]⁺.

-   (S)-(3-bromo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine    hydrochloride: ¹H NMR (300 MHz, DMSO-d₆): δ 8.40 (s, 3H), 7.63-7.61    (d, 1H), 6.92-6.89 (d, 1H), 5.50-5.48 (d, 1H), 4.68 (m, 1H),    4.35-4.22 (m, 3H), 3.60 (m, 1H), 3.00 (m, 1H).-   (R)-(3-bromo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine    hydrochloride: ¹H NMR (300 MHz, DMSO-d₆): δ 8.30 (s, 3H), 7.64-7.61    (d, 1H), 6.93-6.90 (d, 1H), 5.51-5.49 (d, 1H), 4.68 (m, 1H),    4.36-4.23 (m, 3H), 3.61 (m, 1H), 3.05-3.01 (m, 1H).

Method B (a) (S)-tert-butyl((3-bromo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate

A mixture of (S)-tert-butyl((7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(110.0 g, 360.50 mmol) (Example 4, Method B, (h)) and NBS (67.4 g,378.53 mmol) in DCE (1.1 L) was heated at 50° C. for 6 h. The solutionwas washed with hot water (1 L) three times and the organic solution wasconcentrated under vacuum to obtain the desired product (132.0 g, crude)as a yellow gum (used in next step without purification). ¹H NMR (400MHz, DMSO-d₆): 7.57-7.55 (d, J=8 Hz, 1H), 6.96 (s, 1H), 6.85-6.83 (d,J=8 Hz, 1H), 5.25 (m, 1H), 4.71-4.69 (m, 1H), 4.34-4.07 (m, 3H),3.76-3.69 (m, 1H), 3.17-3.16(m, 1H), 1.33 (s, 9H). LC-MS: [M−55]=327.8.

(b) Title Compound

A solution of (S)-tert-butyl((3-bromo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(130.0 g, crude) and conc. HCl (100 mL) in 1,4-dioxane (500 mL) wasstirred at r.t. for 8 h, during which time colorless solids wereprecipitated and filtered and washed with 2-propanol (200 mL). The solidwas dried under vacuum at 50° C. for 6 h to obtain the hydrochloridesalt of desired product (60.0 g, 51.9% total yield over two steps) as acolorless solid. ¹H NMR (400 MHz, DMSO-d₆): 8.45 (s, 3H), 7.64-7.62 (d,J=8, 1H), 6.92-6.90 (d, J=8, 1H), 5.52 (m, 1H), 4.69 (m, 1H), 4.37-4.15(m, 3H), 3.74-3.50 (m, 1H), 3.05-2.95 (m, 1H). ¹³C NMR (400 MHz,DMSO-d₆): 161.80, 151.28, 137.57, 118.64, 107.18, 80.04, 73.86, 69.18,41.88. LC-MS: [M+1]⁺=283.9.

Example 33-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride (G3-Cl)

(a)3-chloro-2-(nitromethyl)-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulene

To a solution of2-(nitromethyl)-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulene (29g, 123.4 mmol) (Example 1, (d)) in DMF (250 mL) at 80° C. was added asolution of NCS (16.5 g, 123.4 mmol) in DMF (100 mL). The mixture wasstirred for 30 min at 80° C. The reaction mixture was poured intoice-water and extracted with EtOAc (200 mL×3). The combined organiclayers were washed with brine, dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The residue was purified byre-crystallization from petroleum ether/ethyl acetate (10:1) to give 24g of crude product. MS (ESI) m/z=270 [M+H]⁺. ¹H NMR (300 MHz, DMSO-d₆):δ 7.52-7.49 (d, 1H), 6.99-6.96 (d, 1H), 5.96-5.93 (m, 1H), 5.42-5.30 (m,1H), 4.80-4.61 (m, 2H), 4.43-4.17 (m, 3H).

(b) Title Compound

A solution of3-chloro-2-(nitromethyl)-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulene(24 g, 89.22 mmol), Raney Ni (4.0 g) and 7 M NH₃ in MeOH (20 mL) inmethanol (300 mL) was shaken under an atmosphere of H₂ for 2 h at rt.The mixture was filtered through a bed of Celite and the filtrate wasconcentrated under vacuum. The crude amine was dissolved in MeOH (20 mL)and concentrated HCl (5 mL) was added. The resulting mixture was stirredat rt for 1 h and then concentrated under reduced pressure. Theresulting solid was washed with acetonitrile/hexanes (2:1, 2×200 mL) togive the desired product (12 g, 50%) as a white solid. ¹H NMR (300 MHz,DMSO-d₆): δ 8.19 (s, 3H), 7.51-7.48 (d, 1H), 6.99-6.96 (d, 1H),5.56-5.54 (d, 1H), 4.69 (m, 1H), 4.36-4.23 (m, 3H), 3.58 (m, 1H),3.05-3.01 (m, 1H). MS (ESI) m/z=240 [M+H]⁺.

Example 4-I(S)-(3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride (G4-Cl)

Method A (a) tert-butyl((3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate

To a mixture of(3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride (8.0 g, 33.7 mmol) (Example 3,(b)) and triethylamine (10.2g, 101.2 mmol) in dichloromethane (250 mL) at 0° C. was addeddi-tert-butyl dicarbonate (11 g, 50.6 mmol) and the mixture was stirredfor 2 h at rt. The reaction was quenched with sat. NaHCO₃ (150 mL) andthe resulting mixture was extracted with EtOAc (2×200 mL). The combinedorganic layers were dried over anhydrous Na₂SO₄ and concentrated undervacuum. The residue was purified by preparative-HPLC using Daisogel 10μC18 column (250×50 mm) and eluted with gradient water/acetonitrile(0.05% TFA) to give the desired product (4.6 g, 47%). MS (ESI) m/z=284[M−56]^(+.)

(b) Title Compound

The racemic compound tert-butyl((3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamatewas separated via SFC (chiral column CHIRALCEL OJ-H) eluted with EtOH(15%) and CO₂ (85%) and the two chiral compounds (S)-tert-butyl((3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(second eluting isomer, RT=2.9 min) and(R)-tert-butyl((3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(first eluting isomer, RT=2.6 min) were obtained. Each of the chiralcompounds (4.6 g, 13.6 mmol) was stirred at rt in 80 mL of saturated HCl(gas) in Et₂O for 1 h and concentrated to dryness (water bathtemperature <30° C.). The residue was triturated with acetonitrile (2×5mL) and the white solid was dried under high vacuum to give the twoproducts(S)-(3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride (1.2 g) and (R)-(3-chloro-7, 8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamine hydrochloride (2.3 g)respectively as white solids. MS (ESI) m/z=240 [M+H]⁺.

(S)-(3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride: ¹H NMR (300 MHz, DMSO-d₆): δ 8.30 (s, 3H), 7.51-7.48 (d,1H), 6.99-6.96 (d, 1H), 5.59-5.57 (d, 1H), 4.68 (m, 1H), 4.36-4.23 (m,3H), 3.58 (s, 1H), 3.03-2.99 (m, 1H).

G4-Cl-(R)(R)-(3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride: ¹H NMR (300 MHz, DMSO-d₆): δ 8.28 (s, 3H), 7.51-7.48 (d,1H), 6.99-6.96 (d, 1H), 5.58-5.56 (d, 1H), 4.69 (m, 1H), 4.36-4.23 (m,3H), 3.59 (m, 1H), 3.05-3.01 (m, 1H).

Method B (a)(Z)-1-(pyridin-2-yl)-N-((1R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene)methanamine

A mixture of (+)-camphor (371 g, 2.44 mol), pyridin-2-ylmethanamine (277g, 2.56 mol) and BF₃.Et₂O (17 g, 0.12 mol) in toluene (3.7 L) wascharged into a 5 L round bottom flask equipped with a Dean Stark trap,reflux condenser, thermometer and nitrogen inlet. The mixture was heatedto reflux with azeotropic removal of water for 20 h. The mixture wascooled to 15° C. and quenched with 5% aqueous sodium bicarbonate (2.5L), the organic phase was separated and washed with water (1.25 L×2),then the mixture was concentrated down to 2 L under vacuum. The residuewas used in next step without purification. ¹H NMR (400 MHz, DMSO-d₆):8.47-8.48 (d, J=4.4 Hz, 1H), 8.77-8.74 (t, J=7.6 Hz, 1H), 7.43-7.41 (d,J=8.0 Hz, 1H), 7.25-7.22 (dd, J=4.8 Hz, 1H), 4.49-4.38 (dd, J=16.4 Hz,2H), 2.46-2.42 (m, 1H), 1.97-1.93 (m, 2H), 1.84-1.79 (m, 1H), 1.71-1.64(m, 1H), 1.33-1.22 (m, 2H), 0.93 (s, 3H), 0.92 (s, 3H), 0.73 (s, 3H).LCMS: [M+H]⁺=243.

(b)(1R)-1,7,7-trimethyl-N-(pyridin-2-ylmethyl)bicyclo[2.2.1]heptan-2-amine

5% Pt/C (40 g) was charged into a 5 L pressure vessel, followed by asolution of(Z)-1-(pyridin-2-yl)-N-((1R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene)methanamine(2.44 mol) in toluene (2 L). The vessel was pressurized with 100 psihydrogen for a period of 12 h. The solid was filtered through Celite®and the cake was washed with toluene (1 L). The filtrate wasconcentrated under vacuum to obtain the desired product (435 g obtained,total yield: 73%, over two steps) as a pale yellow oil. ¹H NMR (400 MHz,DMSO-d₆): 8.49-8.48 (d, J=4.8 Hz, 1H), 7.75-7.71 (t, J=7.6 Hz, 1H),7.40-7.38 (d, J=7.6 Hz, 1H), 7.24-7.21 (dd, J=5.2 Hz, 1H), 3.79-3.64(dd, J=14.4 Hz, 2H), 2.53-2.49 (m, 1H), 1.99 (s, 1H), 1.68-1.42 (m, 5H),1.05 (s, 3H), 0.87 (s, 3H), 0.78 (s, 3H), LCMS: [M+H]⁺=245.

(c) 3-(2-(benzyloxy)ethoxy)benzaldehyde

To a solution of 3-hydroxybenzaldehyde (2.90 kg, 23.75 mol), and((2-bromoethoxy)methyl)benzene (4.26 kg, 19.79 mol) in DMF (9.3 L) wasadded K₂CO₃ (3.83 kg, 27.70 mol). The reaction mixture was stirred atr.t. for 24 h. Water (15 L) and tert-butyl methyl ether (23 L) wereadded to the reaction mixture. The organic phase was separated andwashed with 1N NaOH (2×15 L) and water (15 L) sequentially, and thenconcentrated to a minimum. Ethanol (23 L) was added and the solution wasconcentrated under vacuum to afford the desired product (4.7 kg, 93%) asa colourless oil. ¹H NMR (400 MHz, DMSO-d₆): 9.98 (s, 1H), 7.55-7.52 (m,2H), 7.46 (s, 1H), 7.36-7.34 (m, 4H), 7.32-7.26 (m, 2H), 4.57 (s, 2H),4.25-4.22 (t, J=4.4 Hz, 2H), 3.80-3.78 (t, J=4.4 Hz, 2H). LCMS:[M+Na]⁺=279.

(d) (S)-1-(3-(2-(benzyloxy)ethoxy)phenyl)-2-nitroethanol

A mixture of copper (II) acetate (167 g, 0.92 mol),(1R)-1,7,7-trimethyl-N-(pyridin-2-ylmethyl)bicyclo[2.2.1]heptan-2-amine(269 g, 1.10 mol) in ethanol (19 L) was stirred at r.t. for 1 h, then asolution of 3-(2-(benzyloxy)ethoxy)benzaldehyde (4.70 kg, 18.34 mol) inethanol (5 L) was added. The reaction mixture was cooled to atemperature range between −30° C. and −40° C., and then nitromethane(9.9 L, 183.40 mol) was added dropwise, keeping the temperature below−30° C., followed by the addition of diisopropylethylamine (285 g, 2.20mol). The reaction was stirred at −30° C. for 24 h, and then quenchedwith trifluoroacetic acid (314 g, 2.75 mol). 1 N HCl (24 L) and TBME (47L) were added to the resulting solution. The separated organic phase waswashed with water (24 L) and then concentrated under vacuum. The residuewas added to a mixture of petroleum ether/ethyl acetate=5:1 (10 L). Thenthe yellow solid was precipitated, and collected by filtration withBuchner funnel and dried under vacuum at 40° C. for 6 h to afford thedesired product (5.00 kg, 86%) as a white solid. ¹H NMR (400 MHz,DMSO-d₆): 7.38-7.25 (m, 6H), 7.03 (s, 1H), 7.01-6.99 (d, J=7.6 Hz, 1H),6.90-6.87 (dd, J=8.0 Hz, 1H), 6.09-6.08 (d, J=5.2 Hz, 1H), 5.26-5.22 (m,1H), 4.86-4.82 (dd, J=12.4 Hz, 1H), 4.57-4.51 (m, 3H), 4.15-4.13 (m,2H), 3.78-3.76 (t, J=4.8 Hz, 2H). LC-MS: [M+Na]⁺=340.

(e) (S)-1-(3-(2-(benzyloxy)ethoxy)phenyl)-2-(dibenzylamino)ethanolhydrochloride

10% Pd/C (800 g) and 10% Pt/C (200 g) were charged to a pressure vessel,followed by a solution of(S)-1-(3-(2-(benzyloxy)ethoxy)phenyI)-2-nitroethanol (5.00 kg, 15.76mol) in ethanol (50 L). The vessel was pressurized with 100 psi hydrogenfor 12 h at r.t.. The solid was filtered through Celite® and the cakewas washed with ethanol (5 L). To the filtrate, K₂CO₃ (4.80 kg, 34.67mol) and benzyl bromide (5.93 kg, 34.67 mol) were added sequentially.The reaction mixture was stirred at r.t. for 24 h. The solid wasfiltered and washed with ethanol (1 L). The filtrate was diluted withwater (20 L) and then heated to 50° C. The solution was stirred at 50°C. for 30 min and then conc. HCl (1.5 L) was added dropwise over 1 h.The mixture was cooled to 0° C. and held at 0° C. for additional 30 min.The product was filtered and washed with 20% aqueous ethanol (1 L) toafford the hydrochloric salt of desired product (5.00 kg, 63% over twosteps) as a colourless solid. ¹H NMR (400 MHz, DMSO-d₆): 10.67 (s, 1H),7.72-7.68 (m, 4H), 7.47-7.45 (m, 6H), 7.38-7.26 (m, 5H), 7.25-7.21 (t,J=7.6 Hz, 1H), 6.86-6.84 (d, J=8.0 Hz, 1H), 6.77 (s, 1H), 6.77-6.75 (d,J=7.2 Hz, 1H), 6.36 (s, 1H), 5.04-5.02 (d, J=9.2 Hz, 1H), 4.58 (s, 2H),4.51-4.38 (m, 4H), 4.09-4.07 (t, J=4.0 Hz, 2H), 3.77-3.75 (t, J=3.2 Hz,2H), 3.13-2.96 (m, 2H). LC-MS: [M+H]⁺=468.

(f)(S)-7-(2-(benzyloxy)ethoxy)-3-((dibenzylamino)methyl)benzo[c][1,2]oxaborol-1(3H)-ol

To a −30° C. solution of(S)-1-(3-(2-(benzyloxy)ethoxy)phenyl)-2-(dibenzylamino)ethanolhydrochloride (3.85 kg, 7.64 mol) in dry toluene (39 L) under N₂atmosphere was added n-BuLi (15.3 L, 38.20 mol) dropwise over 6 h. Afteraddition, the mixture was stirred at −30° C. for another 1 h, and thencooled to −70° C.; trimethyl borate (3.97 kg, 38.20 mol) was addeddropwise keeping the temperature below −60° C. After addition, thereaction mixture was allowed to warm to r.t. and stirred overnight. Thereaction was quenched with 5% aqueous NaHCO₃ (20 L) and stirredvigorously for 15 min, the resulting suspension was filtered and thefiltrate was separated. The organic layer was washed with water (20 L×3)and concentrated under vacuum and the residue was purified by gelchromatography eluting with petroleum ether/ethyl acetate=5:1 to afforddesired product (1.80 kg, 48%) as a yellow solid. ¹H NMR (400 MHz,DMSO-d₆): 8.81 (s, 1H), 7.39-7.22 (m, 16H), 6.82-6.80 (d, J=7.6 Hz, 1H),6.72-6.70 (d, J=7.6 Hz, 1H), 5.34-5.31 (dd, J=7.6 Hz, 1H), 4.60 (s, 2H),4.22-4.19 (t, J=4.4 Hz, 2H), 3.80-3.72 (m, 6H), 2.88-2.84 (dd, J=13.6Hz, 1H), 2.47-2.45 (dd, J=10 Hz, 1H). LC-MS: [M+H]⁺=494.

(g)(S)-(7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride

10% Pd/C (180 g) was charged to a pressure vessel, followed by asolution of(S)-7-(2-(benzyloxy)ethoxy)-3-((dibenzylamino)methyl)benzo[c][1,2]oxaborol-1(3H)-ol(1.80 kg, 3.65 mol) in methanol (18 L), toluene (3.6 L) and 1 N HCl (4L). The vessel was pressurized with 100 psi hydrogen for a period of 12h at 50° C. The solid was filtered through Celite and the cake waswashed with methanol (1 L). The filtrate was concentrated under vacuumand the residue was treated with 2-propanol (3.6 L), stirred at r.t. for30 min. The resulting solid was collected by filtration and washed with2-propanol (500 mL), dried under vacuum at 50° C. for 6 h to afford thedesired product (680 g, 77%) as a pale yellow powder. ¹H NMR (400 MHz,DMSO-d₆): 8.38 (s, 3H), 7.52-7.48 (t, J=8.0 Hz, 1H), 7.17-7.15 (d, J=7.6Hz, 1H), 6.92-6.90 (d, J=7.6 Hz, 1H), 5.55 (m, 1H), 4.71-4.68 (m, 1H),4.38-4.22 (m, 3H), 3.53-3.50 (m, 1H), 2.91-2.86 (m, 1H). LC-MS:[M+H]⁺=206.

(h) (S)-tert-butyl((7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate

To a solution of(S)-(7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride (390 g, 1.62 mol) and Et₃N (163.4 g, 4.85 mol) in DCM (4.6L) was added (Boc)₂O (353.0 g 1.62 mol) dropwise over 2 h at r.t.. Afteraddition, the reaction mixture was stirred at r.t. for another 3 h. Thereaction was quenched with 1N HCl (4 L) and the organic phase wasseparated and washed with water (4 L), concentrated under vacuum toobtain desired product (460 g, 93%) as a pale white solid. ¹H NMR (400MHz, DMSO-d₆): 7.46-7.42 (t, J=7.6 Hz, 1H), 7.07 (s, 1H), 7.02-7.00 (d,J=7.2 Hz, 1H), 6.87-6.85 (d, J=8.0 Hz, 1H), 5.27 (m, 1H), 4.68-4.65 (m,1H), 4.34-4.18 (m, 3H), 3.41(s, 1H), 3.14-3.08 (m, 1H), 1.38 (s, 9H).LC-MS: [M−55]=250.

(i) (S)-tert-butyl((3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate

A mixture of (S)-tert-butyl((7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(315.0 g, 1.03 mol) and NCS (144.5 g, 1.08 mol) in dichloroethane (3.5L) was heated at 50° C. for 24 h. The solution was washed with hot water(50° C., 4 L×3) and the organic phase was concentrated under vacuum toobtain desired product (400.0 g, crude) as a yellow solid, which wasused in the next step without further purification. ¹H NMR (400 MHz,DMSO-d₆): 7.44-7.42 (d, J=8.4 Hz, 1H), 6.99 (s, 1H), 6.91-6.89 (d, J=8.4Hz, 1H), 5.33 (m, 1H), 4.72-4.69 (m, 1H), 4.35-4.19 (m, 3H), 3.73-3.71(m, 1H), 3.17-3.15(m, 1H), 1.33 (s, 9H). LC-MS: [M−55]=284.

(j) Title Compound

A solution of (S)-tert-butyl((3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(400.0 g, crude) and conc. HCl (500 mL) in 1,4-dioxane (2 L) was stirredat r.t. for 8 h, during which time colourless solids were precipitated,collected and washed with 2-propanol (200 mL). The solid wasrecrystallized from H₂O and dioxane (400 mL/2000 mL) to obtain thehydrochloride salt of desired product (110.0 g, 39%, over two steps). ¹HNMR (400 MHz, DMSO-d₆): 8.48-8.35 (br, 3H), 7.52-7.50 (d, J=8.8 Hz, 1H),7.00-6.97 (d, J=8.4 Hz, 1H), 5.60 (m, 1H), 4.71 (m, 1H), 4.38-4.21 (m,3H), 3.64-3.55 (m, 1H), 3.04-2.99 (m, 1H). ¹³C NMR (400 MHz, DMSO-d6):161.22, 149.15, 134.61, 119.35, 118.31, 79.14, 73.92, 69.22, 41.88.LC-MS: [M+H]⁺=240.

Example 4-II(S)-(3-chloro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminedihydrogensulfate.H₂O (G4-Cl)

A mixture of (S)-tert-butyl((7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(13.25 kg) and NCS (8.75 kg) in dichloroethane (132.5 L) was heated at70° C. until the reaction judged complete by HPLC. The mixture wasconcentrated under reduced pressure, cooled to 25° C. and acetone (106L) added. The slurry was filtered, washing with acetone (26.5 L). Thewet cake was slurried in water (13.25 L) and 1,4-dioxane (66.25 L),heated to 50° C. for 20-30 minutes, cooled to 15° C., filtered and thecake washed with 1,4-dioxane (26.5 L). The wet cake was dissolved inmethanol (68.9 L), filtered and the filtrate concentrated under reducedpressure. Methyl tertiary butyl ether (66.25 L) was added to the residueand the mixture concentrated under reduced pressure. Methyl tertiarybutyl ether (78.7 L), isopropanol (8.7 L) and sulphuric acid (4.6 L)were added, the mixture heated to 50° C. and stirred until the sulphatecontent was 24.32-29.72%. The mixture was cooled to 25° C., stirred for1 hour, filtered, the cake washed with methyl tertiary butyl ether (17.5L) and dried to give the desired product (42%).

Example 5(3-fluoro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride) (G5-F)

(a) 1-(2-(benzyloxy)ethoxy)-2-bromo-4-fluorobenzene

A solution of 2-bromo-4-fluorophenol (1.91 g, 10 mmol),((2-bromoethoxy)methyl)benzene (2.6 g, 12 mmol) and K₂CO₃ (2.76 g, 20mmol) in 40 mL of DMF was stirred at 25° C. for 16 h. Then the mixturewas poured into 300 mL of water, extracted with ethyl acetate (200 mL),washed with water (200 mL) and brine (100 mL), and dried over anhydroussodium sulfate. The solvent was evaporated at 40° C. under reducedpressure and the residue was purified by silica gel chromatographyeluting with ethyl acetate and petroleum ether (1:5) to afford theproduct (3.1 g, 95%) as a colorless oil. ¹H NMR (300 MHz, DMSO-d₆): δ7.55 (dd, 1H), 7.36-7.15 (m, 7H), 4.60 (s, 2H), 4.22-4.19 (m, 2H),3.80-3.77 (m, 2H).

(b) 3-(2-(benzyloxy)ethoxy)-2-bromo-6-fluorobenzaldehyde

A solution of 1-(2-(benzyloxy)ethoxy)-2-bromo-4-fluorobenzene (1.6 g,4.9 mmol) in 30 mL of THF was cooled to −70° C., and LDA (2.0 M in THF,3.5 mL, 7 mmol) was added dropwise. The resulting mixture was keptstirring for 2 h at low temperature before a solution of DMF (1.1g, 15mmol) in THF (3 mL) was added. The mixture was stirred for 1 h and thenallowed to warm to 0° C. It was quenched by saturated aq. NH₄Cl and themixture was extracted with ethyl acetate (100 mL). The organic layer waswashed with water (50 mL) and brine (50 mL), and dried over anhydroussodium sulfate. The solvent was removed under reduced pressure and theresidue was purified by silica gel chromatography eluting with ethylacetate and petroleum ether (1:3) to afford the product (1.2 g, 69%) asa colorless oil. ¹H NMR (300 MHz, DMSO-d₆): δ 10.22 (s, 1H), 7.48-7.27(m, 7H), 4.60 (s, 2H), 4.29-4.26 (m, 2H), 3.82-3.79 (m, 2H).

(c) 6-(2-(benzyloxy)ethoxy)-3-fluoro-2-formylphenylboronic acid

A solution of 3-(2-(benzyloxy)ethoxy)-2-bromo-6-fluorobenzaldehyde (1 g,2.8 mmol), Pin₂B₂ (1 g, 4 mmol), KOAc (0.56 g, 6 mmol) and Pd(dppf)Cl₂(0.05 g) in 30 mL of THF was degassed with N₂ for six times. Then themixture was heated at 100° C. (microwave irradiated) for 4 h. Thereaction mixture was cooled to room temperature, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel chromatography eluting with ethyl acetate and petroleum ether (1:5).The fractions were combined and concentrated under reduced pressure. Theresidue was dissolved in THF (20 mL) and 6N HCl (4 mL) and the resultingmixture was stirred at room temperature for 1 h. After it was extractedwith ethyl acetate (20 ml×3), the combined organic layer wasconcentrated under reduced pressure to afford the crude product (0.5 g,56%). It was used directly in the next step without furtherpurification. LC-MS: 336.0 [M+H₂O]⁺.

(d)7-(2-(benzyloxy)ethoxy)-4-fluoro-3-(nitromethyl)benzo[c][1,2]oxaborol-1(3H)-ol

To a stirred solution of6-(2-(benzyloxy)ethoxy)-3-fluoro-2-formylphenylboronic acid (0.5 g, 1.6mmol) and CH₃NO₂ (0.2 g, 3.5 mmol) in 10 mL of THF was added a solutionof NaOH (0.028 g, 0.7 mmol) in 3 mL of water at room temperature. Thenthe mixture was stirred at room temperature for 16 h and acidified withconc. HCl to pH=1 at 0° C. The mixture was extracted with ethyl acetate(20 mL) and the organic layer was washed with water (10 mL) and brine(10 mL) then dried over anhydrous sodium sulphate. After the solvent wasremoved under reduced pressure, the residue was purified by silica gelchromatography eluting with ethyl acetate and petroleum ether (1:10) toafford the crude product (0.5 g, 88%) as a colourless oil. LC-MS: 379.0[M+H₂O]⁺.

(e) Title Compound

A solution of7-(2-(benzyloxy)ethoxy)-4-fluoro-3-(nitromethyl)benzo[c][1,2]oxaborol-1(3H)-ol(0.5 g, 1.4 mmol) and Pd/C (10%, 0.1 g) in 20 mL of methanol washydrogenated under 1 atm of H₂ at room temperature for 48 h. Then it wasfiltered through a bed of Celite and the filtrate was concentrated underreduced pressure to give an oil. The crude product was purified bypreparative-HPLC using Daisogel 10μ C18 column (250×50 mm) and elutedwith a gradient of water/acetonitrile (0.05% TFA). The collectedfraction was concentrated under reduced pressure. The residue wasdissolved in ether (5 mL) and 2N HCl (0.2 mL) was added. The resultingmixture was stirred at room temperature for 1 h. The solid was collectedby filtration and washed with ether (10 mL) to give the title compound(0.035 g, 10%) as a white solid. LC-MS: 223.9 [M+H]⁺. ¹H NMR (400 MHz,DMSO-d₆): δ 8.22 (brs, 3H), 7.33 (t, 1H), 6.97 (dd, 1H), 5.68 (d, 1H),4.69 (brs, 1H), 4.37-4.23 (m, 3H), 3.43-3.40 (m, 1H), 3.03 (t, 1H).

Example 6(S)-(3-iodo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride (G6-I)

(a) (S)-tert-butyl((3-iodo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate

A solution of (S)-tert-butyl((7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(300 mg, 0.98 mmol) (Example 4, Method B, (h)) and NIS (265 mg, 1.18mmol) in 6 mL of AcOH was stirred at room temperature for 24 h. Thesolvent was evaporated at 40° C. under reduced pressure. The residue waspurified by preparative-HPLC using Daisogel 10μ C18 column (250×50 mm)and eluted with a gradient of water/acetonitrile (0.05% TFA) to affordthe product (200 mg, 47%) as light yellow oil. LC-MS: 432 [M+H]⁺.

(b) Title Compound

A solution of (S)-tert-butyl((3-iodo-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(140 mg, 0.32 mmol) and TFA (0.5 ml) in 5 mL of DCM was stirred at roomtemperature for 2 h. The solvent was evaporated at 40° C. under reducedpressure. The residue was dissolved in ether (5 mL) and 2N HCl in water(0.2 mL) was added. The resulting mixture was stirred at roomtemperature for 15 min. The solid was collected by filtration and washedwith ether (10 mL) to give the title compound (90 mg, 75%) as a whitesolid. LC-MS: 332.0 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 8.47 (brs, 3H),7.80 (d, 1H), 6.78 (d, 1H), 5.37 (m, 1H), 4.72-4.53 (m, 1H), 44⁹-4.08(m, 3H), 3.78-3.5₁ (m, 1H), 3.06-2.78 (m, 1H).

Example 7(3-chloro-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride (G7-Cl)

(a) 2-bromo-3-(2-hydroxypropoxy)benzaldehyde

A solution of 2-bromo-3-hydroxybenzaldehyde (6.0 g, 29.85 mmol),1-chloropropan-2-ol (8.46 g, 89.55 mmol) and K₂CO₃ (8.24, 59.7 mmol) inDMF (100 mL) was stirred at 100° C. overnight. Then the reaction mixturewas quenched by adding water (4 L) and then extracted with EtOAc (3×1.5L). The combined organic layers were washed with brine (250 mL), driedover anhydrous Na₂SO₄ and concentrated to dryness in vacuo. The residuewas purified by column chromatography on silica gel (petroleumether:ethyl acetate=5:1 to 2:1) to give the target crude compound (8.77g). MS (ESI) m/z=259/261 [M+H]⁺.

(b)3-(2-hydroxypropoxy)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde

A solution of 2-bromo-3-(2-hydroxypropoxy)benzaldehyde (8.77 g, 34 mmol)4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (17.27 g, 68mmol), Pd(dppf)Cl₂ (2.49 g, 3.4 mmol) and KOAc (9.99 g, 102 mmol) indioxane (200 mL) was stirred at 100° C. overnight. Then the reactionmixture was quenched by adding water (200 mL) and then extracted withEtOAc (3×200 mL). The combined organic layers were washed with brine(250 mL), dried over anhydrous Na₂SO₄ and concentrated to dryness invacuo. The residue was purified by column chromatography on silica gel(petroleum ether:ethyl acetate=5:1 to 1:1) to give the target crudecompound (6 g). MS (ESI) m/z=307 [M+H]⁺.

(c)8-methyl-2-(nitromethyl)-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulene

To a solution of NaOH (261.4 mg, 6.54 mmol) in water (8 mL) was addednitromethane (1.2 g, 19.6 mmol) at 5-10° C. After stirring for 15 min at5-10° C., CTAB (0.19 g, 0.52 mmol) was added to the reaction mixture andfollowed by the addition of3-(2-hydroxypropoxy)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde(2.0 g, 6.54 mmol) at 5-10° C. The reaction mixture was stirred at rtfor 5 h. The reaction mixture was acidified to pH=1 using dilutedhydrochloric acid and stirred at rt overnight. The reaction mixture wasfiltered to give the target compound (541 mg, 33%).

(d)(8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamineacetate

A solution of8-methyl-2-(nitromethyl)-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulene(541 mg, 2.173 mmol) and palladium hydroxide (300 mg) in acetic acid (10mL) was shaken under an atmosphere of H₂ overnight at room temperature.The mixture was filtered through a bed of Celite and the filtrate wasconcentrated in vacuo to give the crude compound (350 mg). MS (ESI)m/z=220 [M+H]⁺.

(e) tert-butyl((8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate

To the mixture of crude compound(8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamineacetate (3.0 g, 10.75 mmol) and triethylamine (6.5 g, 64.5 mmol) indichloromethane (100 mL) at 0° C. was added di-tert-butyl dicarbonate(3.5 g, 16.13 mmol) and the mixture was stirred for 2 h at roomtemperature. The reaction was quenched with sat. NaHCO₃ (15 mL) and theresulting mixture was extracted with EtOAc (3×80 mL), the combinedorganic layers were dried over anhydrous Na₂SO₄ and concentrated invacuo. The residue was purified by preparative-HPLC using a Daisogel 10μC18 column (250×50 mm), eluted with gradient water/acetonitrile (0.05%TFA) to give the product (700 mg). MS (ESI) m/z=264 [M−56]⁺. ¹H NMR (300MHz, DMSO-d₆): δ 7.44-7.39 (m, 1H), 7.01-6.98 (m, 2H), 6.88-6.85 (m,1H), 5.24 (m, 1H), 4.52-4.44 (m, 2H), 4.18-4.00 (m, 1H), 3.39-3.36 (m,1H), 3.15-3.06 (m, 1H), 1.42-1.09 (m, 15H).

(f) tert-butyl((3-chloro-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate

To a solution of tert-butyl((8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(300 mg, 0.94 mmol) and 1-chloropyrrolidine-2,5-dione (151.4 mg, 1.13mmol) in CH₃CN (20 mL) was added 2,2′-Azobis(2-methylpropionitrile (15.4mg, 0.094 mmol) and the mixture was stirred for 2 h at 90° C. Thereaction mixture was then concentrated under high vacuum and the residuewas purified by preparative-HPLC using a Gemini® 5μ C18 column (150×21.2mm) and eluted with gradient water/acetonitrile (0.05% TFA) to give thedesired product (150 mg, 45%). MS (ESI) m/z=298 [M−56]⁺.

(g) Title Compound

tert-butyl((3-chloro-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(150 mg, 0.425mmol) in Et₂O/HCl and Et₂O (10 mL) was stirred at roomtemperature for 2 h and concentrated to dryness (water bath <30° C.).The residue was washed with acetonitrile (2×5 mL) and the white solidwas dried in high vacuo to give the product (120 mg, 97%) as a whitesolid. ¹H NMR (300 MHz, DMSO-d₆): δ 8.28 (s, 3H), 7.51-7.48 (d, 1H),7.02-6.99 (d, 1H), 5.58-5.56 (d, 1H), 4.57 (m, 2H), 4.33-4.17 (m, 1H),3.72-3.56 (m, 1H), 3.05-3.01 (m, 1H), 1.30-1.23 (m, 3H). MS (ESI)m/z=254 [M+H]⁺.

Example 8

(3-bromo-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride (G8-Br)

(a) tert-butyl((3-bromo-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate

To a solution of tert-butyl((8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(180 mg, 0.564 mmol) (Example 5, (e)) and 1-bromopyrrolidine-2,5-dione(120 mg, 0.677 mmol) in CH₃CN (20 mL) was added2,2′-Azobis(2-methylpropionitrile (9.2 mg, 0.056 mmol) and the mixturewas stirred for 2 h at 90° C. The reaction mixture was then concentratedin high vacuo and the residue was purified by preparative-HPLC using aGemini® 5u C18 column (150×21.2 mm) eluted with gradientwater/acetonitrile (0.05% TFA) to give the product (60 mg). MS (ESI)m/z=342/344 [M−56]⁺.

(b) Title Compound

tert-butyl((3-bromo-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(60 mg, 0.15 mmol) in saturated HCl (gas) in Et₂O (10 mL) was stirred atrt for 2 h and concentrated to dryness (water bath temperature <30° C.).The residue purified by preparative-HPLC using a Gemini® 5u C18 column(150×21.2 mm) eluted with gradient water/acetonitrile (0.05% TFA) togive the product (20 mg) as a white solid. ¹H NMR (400 MHz, DMSO-d₆): δ8.12 (br, 3H), 7.65 (m, 1H), 6.96 (m, 1H), 5.45 (m, 1H), 4.58 (m, 2H),4.29-4.16 (m, 1H), 3.77-3.59 (m, 1H), 3.04 (m, 1H), 1.29-1.21 (d, 3H).MS (ESI) m/z=298/300 [M+H]⁺.

Example 9(3-bromo-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride (G9-Br)

(a) 2-bromo-3-(2-hydroxy-2-methyl propoxy)benzaldehyde

A solution of 2-bromo-3-hydroxybenzaldehyde (7.5 g, 37.3 mmol),1-chloro-2-methylpropan-2-ol (9.4 g, 85.6 mmol) and Na₂CO₃ (6.7 g, 63.2mmol) in 70 mL of DMSO was stirred at 140° C. for 3 hours. Then themixture was cooled to room temperature, poured into 300 mL of water,extracted with ethyl acetate (600 mL), washed with water (300 mL), brine(50 mL), dried over anhydrous sodium sulfate. The solvent was evaporatedat 40° C. under reduced pressure and the residue was purified by silicagel chromatography, eluting with a mixture of ethyl acetate andpetroleum ether (1:3) to give the title compound (9.2 g, 90.3%) as acolorless oil. ¹H NMR (300 MHz, CDCl₃): δ 10.43 (s, 1H), 7.54 (dd, 1H,J1=3.0, J2=7.5), 7.40˜7.34 (m, 1H), 7.54 (dd, 1H, J1=3, J2=7.5), 3.90(s, 2H), 1.42 (s, 6H).

(b)3-(2-hydroxy-2-methylpropoxy)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde

A solution of 2-bromo-3-(2-hydroxy-2-methylpropoxy)benzaldehyde (9.2 g,33.7 mmol), Pin₂B₂ (17.1 g, 67.4 mmol), KOAc (9.9 g, 101.1 mmol) andPd(dppf)Cl₂ (2.5 g) in 240 mL of 1,4-dioxane was degassed with N₂ forsix times. Then the reaction was stirred at 99° C. under nitrogen for 16hours. The reaction was cooled, filtered, then evaporated at 40° C.under reduced pressure and the residue was purified by silica gelchromatography, eluting with a mixture of ethyl acetate and petroleumether (1:5) to give the title compound (10 g, crude) including de-Brby-product (used directly in the next step without furtherpurification).

(c)8,8-dimethyl-2-(nitromethyl)-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulene

To a stirred solution of3-(2-hydroxy-2-methylpropoxy)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde(10 g, 31.3 mmol) and CH₃NO₂ (5.7 g, 93.8 mmol) in 100 mL of THF wasadded a solution of NaOH (1.25 g, 31.3 mmol) in 60 mL of water at roomtemperature. Then the reaction was stirred at room temperature for 16hours. Then the reaction was acidified by conc. HCl to pH=1 at 0° C. andstirred at room temperature for 1 hour. The mixture was extracted withethyl acetate (100 mL), washed with water (30 mL), then brine (30 mL),dried over anhydrous sodium sulphate. The solvent was evaporated at 40°C. under reduced pressure and the residue was purified by silica gelchromatography eluting with a mixture of ethyl acetate and petroleumether (1:10) to give the title compound (3 g, 36.5%) as a colourlessoil.

(d)(8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamineacetate

A solution of8,8-dimethyl-2-(nitromethyl)-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulene(1 g, 3.8 mmol) and Pd(OH)₂ (10%, 0.2 g) in 20 mL of acetic acid washydrogenated at 1 atm of H₂ at rt for 16 hours. Then the mixture wasfiltered and the solvent was evaporated at 40° C. under reduced pressureto give the title compound (0.9 g, crude) as an oil (acetate salt).LC-MS: 234.1 [M+H]⁺.

(e) tert-butyl((8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate

To a stirred solution of(8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanamineacetate (0.7 g, 2.39 mmol) in 70 mL of CH₂Cl₂ cooled to 0° C. was addedEt₃N (0.61 g, 6.0 mmol). Then Boc₂O (0.98 g, 4.5 mmol) was added in oneportion, and the reaction was stirred at room temperature for 16 hours.The mixture was washed with 0.3 N HCl (30 mL), water (30 mL) and driedover anhydrous sodium sulphate. The solvent was evaporated at 40° C. atreduced pressure and the residue was purified by silica gelchromatography eluting with a mixture of ethyl acetate and petroleumether (1:4) to give the title compound (0.63 g, 79%) as an oil. LC-MS:234.1 [M+H-100]⁺.

(f) tert-butyl((3-bromo-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate

A solution of tert-butyl((8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(232 g, 0.70 mmol), NBS (143 mg, 0.80 mmol) and AIBN (20 mg) in 30 mL ofacetonitrile was stirred at reflux for 1 hour. The solvent wasevaporated at 40° C. at reduced pressure and the residue was purified bysilica gel chromatography eluting with a mixture of ethyl acetate andpetroleum ether (1:4) to give the title compound (260 mg, 88.6%) as asolid. LC-MS: 312.0/314.0 [M+H-100]⁺.

(g) Title Compound

A solution of tert-butyl((3-bromo-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(260 mg, 0.63 mmol) in a saturated HCl solution in 1,4-dioxane (20 mL)was stirred at room temperature for 3 hours. The solvent was evaporatedat 40° C. under reduced pressure and the residue was purified bypreparative-HPLC using a Gemini® 5u C18 column (150×21.2 mm) eluted withgradient water/acetonitrile (0.05% TFA) treating with 0.1 mL ofconcentrated HCl to give the desired product (20 mg, 9.1%) as a whitesolid. LC-MS: 311.9 [M+H]⁺. ¹H NMR (400 MHz, CD₃OD): δ 7.63 (d, 1H,J=8), 6.95 (d, 1H, J=8), 5.52˜5.45 (m, 1H), 4.41 (d, 1H, J=12), 4.17 (d,1H, J=16), 4.09-3.85 (m, 1H), 3.13-2.98 (m, 1H), 1.37-1.30 (m, 6H).

Example 10(S)-(3-bromo-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride (G10-Br);

(a) (S)-tert-butyl((3-bromo-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate

A solution of tert-butyl((8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(5.5g, 16.5 mmol) (Example 9, (e)) and NBS (3.2 g, 18.2 mmol) in 100 mLof dichloroethane was stirred at 50° C. for 18 hours. The solvent wasevaporated at 40° C. under reduced pressure and the residue was purifiedby silica gel chromatography eluting with a mixture of ethyl acetate andpetroleum ether (1:10) to give the title compound (5.9 g, 86.5%) as anoil. The racemic compound separated via SFC (chiral column CHIRALPAKAD-H, eluted with EtOH (20%) and CO₂ (80%)) to give 2.2g of (S)-isomer(first eluting isomer, RT=3.0 min) and 2.2 g of (R)-isomer (secondeluting isomer, RT=4.1 min). LC-MS: 312.0/314.0 [M+H-100]⁺.

(b) Title Compound

Dry HCl was bubbled through a solution of (S)-tert-butyl((3-bromo-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(2.2 g, 5.34 mmol) in diethyl ether (150 mL) at room temperature for 3hours and then stirred for 18 hours. The solvent was filtered and thefilter cake was dried in vacuo to give the (S)-isomer (1.4 g, 76%) as awhite solid. LC-MS: 311.9 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 8.23(brs, 3H), 7.64 (d, 1H, J=8), 6.96 (d, 1H, J=8), 5.48-5.46 (m, 1H),4.43-4.40 (m, 1H), 4.21-4.10 (m, 1H), 3.75-3.55 (m, 1H), 3.05-2.95 (m,1H), 1.36-1.27 (ds, 6H). Similarly, the acid treatment of (R)-tert-butyl((3-bromo-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamategave the corresponding (R)-isomer as a white solid (1.4 g, 76%). LC-MS:312.0 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 8.29 (brs, 3H), 7.65 (d, 1H,J=8), 6.96 (d, 1H, J=8), 5.48-5.46 (m, 1H), 4.42-4.39 (m, 1H), 4.22-4.10(m, 1H), 3.75-3.50 (m, 1H), 3.03-2.93 (m, 1H), 1.36-1.27 (ds, 6H).

Example 11 (3-chloro-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride (G11-Cl)

(a) tert-butyl((3-chloro-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate

A solution of tert-butyl((8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(519 mg, 1.56 mmol) (Example 9, (e)), NCS (250 mg, 1.87 mmol) and AIBN(30 mg) in 50 mL of acetonitrile was stirred at reflux for 1 hour. Thesolvent was evaporated at 40° C. under reduced pressure and the residuewas purified by silica gel chromatography eluting with a mixture ofethyl acetate and petroleum ether (1:5) to afford the desired product(300 mg, 52.4%, containing 6-Cl isomer) as a solid. LC-MS: 268.1[M+H-100]⁺.

(b) Title Compound

A solution of tert-butyl((3-chloro-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(300mg, 0.82 mmol) in a saturated HCl solution in 1,4-dioxane (30 mL)was stirred at room temperature for 3 hours. The solvent was evaporatedat 40° C. under reduced pressure and the residue was purified bypreparative-HPLC using a Gemini® 5u C18 column (150×21.2 mm) eluted withgradient water/acetonitrile (0.05% TFA) followed by treating with 0.1 mLof conc. HCl to give the desired product (94 mg, 37.9%) as a whitesolid. LC-MS: 268.1 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 8.40 (brs, 3H),7.52 (d, 1H, J=8), 7.02 (d, 1H, J=8), 5.60-5.58 (m, 1H), 4.42˜4.38 (m,1H), 4.23˜4.07 (m, 1H), 3.67˜3.57 (m, 1H), 3.02˜2.92 (m, 1H), 1.36˜1.27(m, 6H).

Example 12(S)-(3-chloro-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methanaminehydrochloride (G12-Cl)

(a) (S)-tert-butyl((3-chloro-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate

A solution of tert-butyl((8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(4.1g, 12.3 mmol) (Example 9, (e)) and NCS (1.73 g, 13 mmol) in 100 mLof dichloroethane was stirred at 50° C. for 5 hours. The solvent wasevaporated at 40° C. under reduced pressure and the residue was purifiedby silica gel chromatography eluting with a mixture of ethyl acetate andpetroleum ether (1:10) to give the title compound (2.6 g, 58%) as anoil. The racemic compound was separated via SFC (chiral column CHIRALPAKAD-H, eluted with EtOH (20%) and CO₂ (80%)) to give 1.2 g of (S)-isomer(first eluting isomer, RT=2.6 min) and 1.2 g of (R)-isomer (secondeluting isomer, RT=3.5 min. LC-MS: 268.0 [M+H-100]⁺.

(b) Title Compound

Dry HCl was bubbled through a solution of (S)-tert-butyl((3-chloro-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(1.2 g, 3.27 mmol) in diethyl ether (150 mL) at room temperature for 3hours and then stirred for 18 hours. The solvent was filtered and thefilter cake was dried in vacuo to give the (S)-isomer (0.8 g, 80%) as awhite solid. LC-MS: 268 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 8.34 (brs,3H), 7.52 (d, 1H, J=8), 7.02 (d, 1H, J=8), 5.58-5.56 (m, 1H), 4.42-4.39(m, 1H), 4.22˜4.07 (m, 1H), 3.67˜3.53 (m, 1H), 3.03˜2.95 (m, 1H),1.36˜1.27 (ds, 6H).

Similarly, the acid treatment of (R)-tert-butyl((3-chloro-8,8-dimethyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate gave thecorresponding (R)-isomer (G25-Cl(R)) as a white solid (1.2 g, 80%).LC-MS: 268 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 8.33 (bs, 3H), 7.52 (d,1H, J=8), 7.02 (d, 1H, J=8), 5.58 (m, 1H), 4.42˜4.39 (m, 1H), 4.21˜4.07(m, 1H), 3.67˜3.54 (m, 1H), 3.03˜2.95 (m, 1H), 1.36˜1.27 (ds, 6H).

Example 13((2S,8R)-2-(aminomethyl)-3-fluoro-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-8-yl)methanolhydrochloride (C15-F)

(a) (S)-5-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-2-fluorobenzaldehyde

A solution of 2-fluoro-5-hydroxybenzaldehyde (1.9 g, 13.6 mmol),(R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl 4-methylbenzenesulfonate (4.3g, 15 mmol) and K₂CO₃ (2.37 g, 17.2 mmol) in 40 mL of DMSO was stirredat 70° C. for 16 h. Then the mixture was poured into 300 mL of water,extracted with ethyl acetate (200 mL), washed with water (200 mL) andbrine (100 mL), and dried over anhydrous sodium sulfate. The solvent wasevaporated at 40° C. under reduced pressure and the residue was purifiedby silica gel chromatography eluting with ethyl acetate and petroleumether (1:5) to afford the product (2.9 g, 84%) as a colorless oil.LC-MS: 255.1 [M+H]⁺. ¹H NMR (300 MHz, CD₃OD): δ 10.30 (s, 1H), 7.31-7.28(m, 1H), 7.16-7.05 (m, 2H), 4.49-4.45 (m, 1H), 4.18-3.85 (m, 4H),1.45-1.40 (d, 6H).

(b)(S)-1-(5-(((S)-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-2-fluorophenyl)-2-nitroethanol

A mixture of copper (II) acetate (0.2 g, 1.1 mmol),(1R)-1,7,7-trimethyl-N-(pyridin-2-ylmethyl)bicyclo[2.2.1]heptan-2-amine(0.3 g, 1.23 mmol) (Example 4, Method B, (b)) in ethanol (30 mL) wasstirred at r.t. for 1 h, then a solution of(S)-5-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-2-fluorobenzaldehyde(2.9 g, 11.4 mmol) in ethanol (50 mL) was added. The reaction mixturewas cooled to −35° C. to −40° C., and then nitromethane (7 g, 115 mmol)was added dropwise, maintaining the temperature below -35° C., followedby the addition of diisopropylethylamine (0.32 g, 2.50 mmol). Thereaction was stirred at −35° C. for 24 h, and then quenched withtrifluoroacetic acid (0.29 g, 2.5 mmol). EtOAc (200 mL) was added to theresulting solution. The separated organic phase was washed with water(200 mL) and then concentrated under vacuum. The residue was purified bysilica gel chromatography eluting with ethyl acetate and petroleum ether(1:10) to afford the product (3.3 g, 92%) as a colourless oil. LC-MS:316.1 [M+H]⁺.

(c)(S)-2-amino-1-(5-(((S)-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-2-fluorophenyl)ethanol

A solution of(S)-1-(5-(((S)-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-2-fluorophenyl)-2-nitroethanol(3.2 g, 10.2 mmol) and Pd/C (10%, 0.5 g) in 70 mL of methanol washydrogenated under 1 atm of H₂ at room temperature for 48 h. Then it wasfiltered through a bed of Celite and the filtrate was concentrated underreduced pressure to afford the crude product (2.9 g, 100%) as acolourless oil. It was used directly in the next step without furtherpurification. LC-MS: 286.2 [M+H]⁺.

(d)(S)-2-(dibenzylamino)-1-(5-(((S)-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-2-fluorophenyl)ethanol

To a stirred solution of(S)-2-amino-1-(5-(((S)-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-2-fluorophenyl)ethanol(2.9 g, 10.2 mmol) in 50 mL of EtOH were added K₂CO₃ (2.8 g, 20.3 mmol)and BnBr (3.6 g, 21 mmol). The reaction mixture was stirred overnight atroom temperature. The solvent was removed under reduced pressure and theresidue was purified by silica gel chromatography eluting with ethylacetate and petroleum ether (1:10) to afford the product (3.8 g, 80%) asa colourless oil. LC-MS: 466.2 [M+H]⁺

(e)(S)-3-((dibenzylamino)methyl)-7-(((S)-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-4-fluorobenzo[c][1,2]oxaborol-1(3H)-ol

To a solution of(S)-2-(dibenzylamino)-1-(5-(((S)-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-2-fluorophenyl)ethanol(3.3 g, 7.1 mmol) in dry toluene (40 mL) at −30° C. under N₂ atmospherewas added n-BuLi (2.5 M in hexane, 20 mL, 50 mmol) dropwise over 30minutes. After addition, the mixture was stirred at 0° C. for another 2h, and then cooled to −70° C.; trimethyl borate (5.2 g, 50 mol) wasadded dropwise keeping the temperature below −50° C. After addition, thereaction mixture was allowed to warm to −40° C. for 3 h and then warmedto r.t. and stirred overnight. The reaction was quenched with 5% aqueousNaHCO₃ (20 mL) and stirred vigorously for 15 min, the resultingsuspension was filtered and the filtrate was separated. The organiclayer was washed with water (20 mL×3) and concentrated in vacuum toafford the crude product (3 g, 86%) as a yellow oil. LC-MS: 492.2 [M+1]⁺

(f) Title Compound

A solution of(S)-3-((dibenzylamino)methyl)-7-(((S)-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-4-fluorobenzo[c][1,2]oxaborol-1(3H)-ol(3 g, 6.1 mmol) and Pd/C (10%, 0.7 g) in 50 mL of methanol with 2 mL ofconc HCl was hydrogenated under 1 atm of H₂ at room temperature for 48h. Then it was filtered through a bed of Celite and the filtrate wasconcentrated at reduced pressure to give an oil. The crude product waspurified by preparative-HPLC using Daisogel 10μ C18 column (250×50 mm)and eluted with a gradient of water/acetonitrile (0.05% TFA). Thecollected fraction was concentrated under reduced pressure. The residuewas dissolved in ether (30 mL) and sat. HCl (g) in ether (30 mL) and themixture was stirred at room temperature for 1 h. The solid was collectedby filtration and washed with ether to give the title compound (0.4 g,23%) as a white solid. LC-MS: 254.2 [M+H]⁺. ¹H NMR (400 MHz, D2O): δ7.20-7.16 (m, 1H), 6.94-6.91 (m, 1H), 5.55-5.53 (m, 1H), 4.17-4.04 (m,3H), 3.70-3.62 (m, 3H), 3.19-3.14 (m, 1H).

Example 14 ((2S, 8R or 2R,8S)-2-(aminomethyl)-3-chloro-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-8-yl)methanol(C16-Cl) Example 15 ((2S, 8S, or 2R,8R)-2-(aminomethyl)-3-chloro-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-8-yl)methanol(G17-Cl)

(a) ((2-methylallyloxy)methyl)benzene

A solution of methallyl alcohol (80 g, 1.1 mol) in THF (100 mL) wasadded dropwise to a suspension of NaH (66 g, 1.65 mol) in THF (800 mL)at 25° C. under argon. After 1 h, a solution of benzyl bromide (207 g,1.2 mol) in THF (100 mL) was added slowly and the reaction mixture wasstirred at room temperature for 12 h. The reaction mixture was quenchedwith saturated NH₄Cl solution (200 mL) and extracted with ethyl acetate(3×200 mL). The combined organic layers were washed with water (100 mL)and brine (100 mL), dried over Na₂SO₄. The solvent was removed underreduced pressure. The residue was distilled to afford the desiredproduct (134 g, 74%) as colorless oil. ¹H NMR (400 MHz, CDCl₃): δ7.40-7.29 (m, 5H), 5.05 (s, 1H), 4.97 (s, 1H), 4.54 (s, 2H), 3.98 (s,2H), 1.82 (s, 3H).

(2-(benzyloxymethyl)-2-methyloxirane

((2-methylallyloxy)methyl)benzene (41.5 g, 256 mmol) was dissolved inDCM (1200 mL) and cooled to 0° C. m-CPBA (69.7 g, 384 mmol) was addedand the mixture was stirred overnight at room temperature for 12 h.After the white precipitate was filtered off, the filtrate was washedwith saturated Na₂CO₃ solution (200 mL), H₂O (200 mL), and brine. Afterthe solvent was removed under reduced pressure, the crude reside waspurified by silica gel chromatography eluting with ethyl acetate andpetroleum ether (1:20) to afford the pure product (20 g, 44%) ascolorless oil. ¹H NMR (400 MHz, CDCl₃): δ 7.40-7.29 (m, 5H), 4.60 (q,J=12.0 Hz, 2H), 3.61 (d, J=11.0 Hz, 1H), 3.48 (d, J=11.0 Hz, 1H), 2.78(d, J=4.9 Hz, 1H), 2.66 (d, J=4.9 Hz, 1H), 1.43 (s, 3H).

3-(3-(benzyloxy)-2-hydroxy-2-methylpropoxy)-2-bromobenzaldehyde

To a solution of (2-(benzyloxymethyl)-2-methyloxirane (26 g, 145.9 mmol)in DMF (700 mL) was added K₂CO₃ (42 g, 304.3 mmol), followed by2-bromo-3-hydroxybenzaldehyde (30 g, 149.3 mmol). The suspension wasstirred at 90° C. for 6 h. The mixture was cooled down to roomtemperature, diluted with brine and extracted with ethyl acetate (200mL×3). The organic solvent was removed under vacuum and the residue waspurified by silica gel chromatography eluting with ethyl acetate andpetroleum ether (1:20) to afford the pure product (27 g, 49%) as lightyellow oil. ¹H NMR (400 MHz, DMSO-d₆): δ 10.29 (s, 1H), 7.512-7.41 (m,3H), 7.31-7.23 (m, 5H), 4.91 (s, 1H), 4.53 (dd, J₁=12.4 Hz, J₂=17.2 Hz,2H), 4.06 (d, J=9.2 Hz, 1H), 3.91 (d, J=9.2 Hz, 1H), 3.54 (d, J=9.3 Hz,1H), 3.47 (d, J=9.3 Hz, 1H), 1.27 (s, 3H).

3-(3-(benzyloxy)-2-hydroxy-2-methylpropoxy)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde

A solution of3-(3-(benzyloxy)-2-hydroxy-2-methylpropoxy)-2-bromobenzaldehyde (21.3 g,56.2 mmol), Pin₂B₂ (28.6 g, 112.4 mmol), KOAc (6.1 g, 61.9 mmol),PdCl₂(dppf) DCM (1.23 g, 1.7 mmol) in DMF (150 mL) was degassed for 3times with nitrogen. The mixture was heated at 90° C. for 16 h. Afterthe reaction was worked up with ethyl acetate and brine, the residue waspurified by silica gel chromatography eluting with ethyl acetate andpetroleum ether (1:20) to afford the desired product (15.3 g, 64%) aslight yellow oil. LC-MS: 367.1 [344+Na]⁺

(3-(benzyloxy)-2-hydroxy-2-methylpropoxy)-3-(nitromethyl)benzo[c][1,2]oxaborol-1(3H)-ol

To an ice-cold solution of3-(3-(benzyloxy)-2-hydroxy-2-methylpropoxy)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde(18.8 g, 44.1 mmol) in THF was added a solution of NaOH (1.76 g, 44.1mmol) in water (100 mL). After stirring for 15 min, CH₃NO₂ (3.3 g, 53mmol) was added and the mixture was stirred at room temperature for 15h. The reaction solution was acidified with AcOH to pH 3-5. Thesuspension was extracted with ethyl acetate (50 mL×3). The combinedorganic layer was evaporated under vacuum, and the residue was purifiedby silica gel chromatography eluting with ethyl acetate and petroleumether (1:10) to afford the pure product (6.8 g, 40%) as colorless oil.LC-MS: 386.0 [M−1]⁻

(2-(aminomethyl)-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-8-yl)methanolacetate

Pd(OH)₂/C (200 mg) was added to a solution of7-(3-(benzyloxy)-2-hydroxy-2-methylpropoxy)-3-(nitromethyl)benzo[c][1,2]oxaborol-1(3H)-ol(1 g, crude) in AcOH (20 mL). The solution was degassed 3 times with H₂,and stirred at room temperature for 12 h. The reaction mixture wasfiltered through Celite, and the filtrate was concentrated under vacuumto afford the crude product (1 g, crude) as yellow solid.

tert-butyl((8-(hydroxymethyl)-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate

NaHCO₃ (437 mg, 5.2 mmol) was added to a solution of(2-(aminomethyl)-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-8-yl)methanolacetate (650 mg, 2.1 mmol) in t-BuOH (10 mL) and H₂O (10 mL) at roomtemperature. After stirring for 15 min, (Boc)₂O (854 mg, 3.9 mmol) wasadded and the reaction mixture was stirred at room temperature for 2 h.The mixture was acidified with AcOH to pH 6-7 and extracted with DCM (30mL×3). Combined organic layers were evaporated under vacuum, and theresidue was purified by silica gel chromatography eluting with ethylacetate and petroleum ether (1:3) to afford the desired product (400 mg,55%) as courses oil. LC-MS: 294.1 [M−55]⁺

tert-butyl((3-chloro-8-(hydroxymethyl)-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyncarbamate

To a solution of tert-butyl((8-(hydroxymethyl)-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(200 mg, 0.57 mol) in ACN (10 mL) was added NCS (77 mg, 0.57 mmol), andthe solution was stirred at 90° C. for 16 h. The reaction was quenchedwith NH₄Cl solution, extracted with ethyl acetate (20 mL×3). The organiclayer was washed with brine, dried over Na₂SO₄, concentrated in vacuum.The crude residue was purified by silica gel chromatography eluting withethyl acetate and petroleum ether (1:3) to afford the crude product (240mg, crude) as yellow oil. LC-MS: 284.1 [283+H]⁺

Title Compounds

tert-butyl((3-chloro-8-(hydroxymethyl)-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(240 mg, crude) was dissolved in a solution of TFA (1 mL) in DCM (10mL). The solution was stirred at room temperature for 1 h, and then wasconcentrated in vacuum. The crude product was purified bypreparative-HPLC using Daisogel 10μ C18 column (250×50 mm) and elutedwith a gradient of water/acetonitrile (0.05% TFA). The collectedfraction was concentrated under reduced pressure to afford the titlecompounds. ((2S, 8R or 2R,8S)-2-(aminomethyl)-3-chloro-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-8-yl)methanolLC-MS: 284.0 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 8.23 (s, 3H), 7.52 (d,J=8.6 Hz, 1H), 7.03 (d, J=8.5 Hz, 1H), 5.56 (dd, J=8.5, 2.1 Hz, 1H),4.55 (s, 1H), 4.15 (s, 1H), 3.59 (s, 1H), 3.45 (s, 2H), 3.04 (s, 1H),1.21 (s, 3H). ((2S, 8S, or 2R,8R)-2-(aminomethyl)-3-chloro-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-8-yl)methanolLC-MS: 284.1 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 8.12 (s, 2H), 7.51 (s,1H), 7.02 (s, 1H), 5.55 (s, 1H), 4.54 (s, 1H), 4.24-3.92 (m, 1H),3.78-3.29 (m, 3H), 3.02 (s, 1H), 1.25 (s, 3H).

Example 16 ((2S, 8R, or 2R,8S)-2-(aminomethyl)-3-bromo-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-8-yl)methanol(C18-Br) Example 17 ((2S, 8S, or 2R,8R)-2-(aminomethyl)-3-bromo-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-8-yl)methanol(G19-Br)

tert-butyl((3-bromo-8-(hydroxymethyl)-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate

To a solution of tert-butyl((8-(hydroxymethyl)-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(200 mg, 0.57 mmol) in ACN (10 mL) was added NBS (102 mg, 0.57 mmol),and the solution was stirred at 90° C. for 1 h. The reaction wasquenched with NH₄Cl solution, extracted with ethyl acetate (20 mL×3).The organic lay was washed with brine, dried over Na₂SO₄, concentratedin vacuum. The crude residue was purified by silica gel chromatographyeluting with ethyl acetate and petroleum ether (1:3) to afford theproduct (230 mg, crude) as pale solid. LC-MS: 328.1 [M−Boc+H]⁺.

Title Compounds

tert-butyl((3-bromo-8-(hydroxymethyl)-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-2-yl)methyl)carbamate(230 mg, crude) was dissolved in a solution of TFA (1 mL) in DCM (10mL). The solution was stirred at room temperature for 1 h, and then wasconcentrated in vacuum. The crude product was purified bypreparative-HPLC using Daisogel 10μ C18 column (250×50 mm) and elutedwith a gradient of water/acetonitrile (0.05% TFA). The collectedfraction was concentrated under reduced pressure to afford the titlecompounds. ((2S, 8R, or 2R,8S)-2-(aminomethyl)-3-bromo-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-8-yl)methanolLC-MS: 328.0 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.10 (s, 3H), 7.65 (d,J=8.3 Hz, 1H), 7.07-6.88 (m, 1H), 5.56-5.39 (m, 1H), 5.36-5.17 (m, 1H),4.61-4.52 (m, 1H), 4.19-4.07 (m, 1H), 3.62 (d, J=11.9 Hz, 1H), 3.51-3.39(m, 2H), 3.04 (s, 1H), 1.18 (s, 3H). ((2S, 8S, or 2R,8R)-2-(aminomethyl)-3-bromo-8-methyl-7,8-dihydro-2H-1,6,9-trioxa-9a-borabenzo[cd]azulen-8-yl)methanolLC-MS: 328.0 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 8.13 (s, 2H), 7.65 (s,1H), 6.98 (s, 1H), 5.47 (s, 1H), 5.26-5.06 (m, 1H), 4.53 (s, 1H),4.19-3.97 (m, 1H), 3.83-3.56 (m, 1H), 3.51-3.26 (m, 2H), 3.01-2.93 (m,1H), 1.25 (s, 3H).

In Vitro Assays Example 18 MIC Determination Against Mycobacteria

The measurement of the Minimum Inhibitory Concentration (MIC) against M.tuberculosis strains for each tested compound was performed in 96-wellflat-bottom, polystyrene microtiter plates in a final volume of 100 uL.Ten two-fold drug dilutions in neat DMSO starting at 50 mM wereperformed. Drug solutions were added to Middlebrook 7H9 medium (Difco)and isoniazid (INH) (Sigma Aldrich) was used as a positive control with2-fold dilutions of INH starting at 160 ug/mL. The inoculum wasstandardized to approximately 1×107 cfu/ml and diluted 1 in 100 inMiddlebrook 7H9 broth (Difco). This inoculum (100 uL) was added to theentire plate but G-12 and H-12 wells were used as blank controls. Allplates were placed in a sealed box to prevent drying out of theperipheral wells and incubated at 37° C. without shaking for six days. AResazurin solution was prepared by dissolving one tablet of Resazurin(Resazurin Tablets for Milk Testing; Ref 330884Y′ VWR International Ltd)in 30 mL of sterile PBS (phosphate buffered saline). Of this solution,25 uL were added to each well. Fluorescence was measured (Spectramax M5Molecular Devices, Excitation 530 nm, Emission 590 nm) after 48 hours todetermine the MIC value.

Example 19 MIC Against Clinical Strains

The BACTEC MGIT 960 System (Becton Dickinson) was used to carry out MICdetermination in clinical isolates (Institute Carlos III) following themanufacturer instructions. The resistance pattern of clinical isolatesis indicated by the following abbreviations H: Isoniazide, R:Rifampicin, T: Ethionamide, S: Streptomycin, E: Ethambutol, Z:Pyrazynamide, K: Kanamycin, A: Amikacin and CP: Capreomycin. Results forcompound EXAMPLE 4 G4-Cl are shown in Tables 1A, 1B, 2A and 2B, andFIGS. 3 and 4. Results for EXAMPLE 2 G2-Br are shown in Tables 2C and2D, and FIG. 4.

Table 1 provides MIC values for EXAMPLE 4 G4-Cl tested against M.tuberculosis Sensitive (A) and Resistant (B) Clinical Isolates

A Strain 362 457 3 356 357 370 137 169 192 199 206 207 208 223 MIC (μM)0.04 0.04 0.04 0.04 0.04 0.04 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08Strain 231 237 247 248 249 250 253 255 256 257 261 265 269 281 MIC (μM)0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08Strain 292 296 311 314 316 317 322 323 324 326 327 328 329 332 MIC (μM)0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08Strain 333 337 358 361 371 385 391 424 440 442 460 481 716 729 MIC (μM)0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08Strain 730 731 733 734 736 737 52 267 374 274 325 705 161 MIC (μM) 0.080.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.16 0.16 0.16 0.31

B Strain 1819 670 330 198 242 409 141 415 330 Resistance HSRZ HRZ RSR HH HR HRT HRT HS MIC (μM) ≤0.02 0.04 0.04 0.08 0.08 0.08 0.08 0.08 0.08Strain 175 709 732 201 202 277 605 123 106 Resistance S S S HRE S HHSERZ HSERZ HR ACp KTACp MIC (μM) 0.08 0.08 0.08 0.08 0.08 0.08 0.160.16 0.16 Strain 562 139 514 1672 167 254 297 192 CR Resistance HSERZHSERZK HSRT HSRZ HSRZ H HSR HS HSERZ KTA TACp MIC (μM) 0.08 0.08 0.080.08 0.08 0.16 0.16 0.31 0.31

FIG. 3 provides a graphical representation of the MIC data in Tables 1Aand 1B for EXAMPLE 4 G4-Cl, plotted as number of strains with aparticular MIC value (y) versus the particular MIC value obtained (x) inμM. As can be seen in FIG. 3, G4-Cl (Example 4) exhibited a MIC value ofless than 1 μM for more than 85 clinical isolate strains of 97 tested(sensitive and resistant), indicating the very good activity of thiscompound against a significant number of M. tuberculosis clinicalisolate strains. The breakdown is a measured MIC of ≤0.2 μM for 1strain; a measured MIC of 0.04 μM for 8 strains; a measured MIC of 0.08μM for 76 strains; a measured MIC of 0.16 μM for 8 strains; and ameasured MIC of 0.31 μM for 3 strains.

Tables 2A and 2B provide MIC values for EXAMPLE 4 G4-Cl tested againstM. tuberculosis Sensitive (A) and Resistant (B) Clinical Isolates

A Strain 137 169 192 199 206 207 208 223 231 237 247 248 MIC (μM) 0.080.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 Strain 249 255261 265 269 281 292 314 316 317 322 323 MIC (μM) 0.08 0.08 0.08 0.080.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 Strain 324 326 327 328 329 332333 358 MIC (μM) 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

B Strain 175 198 242 330 141 Resistance S H H HSR HRT MIC (μM) 0.08 0.080.08 0.08 0.08 Strain 1819 1672 167 139 123 Resistance HSRZ HSRZ HSRZHSERZKTACp HSERZKTACp MIC (μM) 0.02 0.08 0.08 0.08 0.16

Tables 2C and 2D provide MIC values for EXAMPLE 2 G2-Br tested againstthe same M. tuberculosis Sensitive (A) and Resistant (B) ClinicalIsolates

C Strain 137 169 192 199 206 207 208 223 231 237 247 248 MIC (μM) 0.080.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 Strain 249 255261 265 269 281 292 314 316 317 322 323 MIC (μM) 0.08 0.08 0.08 0.080.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 Strain 324 326 327 328 329 332333 358 MIC (μM) 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

D Strain 175 198 242 330 141 Resistance S H H HSR HRT MIC (μM) 0.08 0.080.08 0.08 0.08 Strain 1819 1672 167 139 123 Resistance HSRZ HSRZ HSRZHSERZKTACp HSERZKTACp MIC (μM) 0.04 0.08 0.08 0.08 0.08

FIG. 4 provides a graphical representation of the MIC data in Tables 2Athrough 2D for G2-Br (Example 2—light bar) and G4-Cl (Example 4—darkbar), plotted as number of strains with a particular MIC value (y)versus the particular MIC value obtained (x), in μM. As can be seen inFIG. 4, G4-Cl (Example 4) and G2-Br (Example 2) exhibited a MIC value ofless than 1 μM for all of but 1 of the 40 M. tuberculosis clinicalisolate strains tested in this experiment. The breakdown is a measuredMIC of ≤0.2 μM for 1 strain (EXAMPLE 4); a measured MIC of 0.04 μM for 1strain (EXAMPLE 2); a measured MIC of 0.08 μM for 40 strains (EXAMPLE 2and EXAMPLE 4); a measured MIC of 0.16 μM for 1 strain (EXAMPLE 2 andEXAMPLE 4); and no measured MIC of 0.31 μM for EXAMPLE 2 or EXAMPLE 4for any strain.

Example 20 General Antimicrobial Activity Assay

Whole-cell antimicrobial activity was determined by broth microdilutionusing the Clinical and Laboratory Standards Institute (CLSI) recommendedprocedure, Document M7-A7, “Methods for Dilution Susceptibility Testsfor Bacteria that Grow Aerobically”.

Table 3 provides MIC values against bacterial strains K12; E. coli K12toIC/Tn10; A. baumannii ATCC 17978; and P. aeruginosa PA01 for compoundsdisclosed in the Examples. As can be seen, the Example compounds do notgenerally possess significant activity across several pathogenic Gramnegative bacteria, as well as an efflux pump deficient E. coli. But asshown in Table 4 below, the compounds disclosed in the Examples dopossess significant activity against M. tuberculosis. Moreover, as canbe seen, tricyclic comparator benzoxaboroles lacking a 4-halogen (egC2-H, C5-H and C12-H) have greater activity against these bacterialstrains whereas tricyclic benzoxaborole compounds with the third ringbeing a seven-membered ring between the 1 and 7 positions of thebenzoxaborole, additionally having 4-halo, 3-aminomethyl substitutionwith (S) stereochemistry at the 3 position (eg G2-Br and G4-Cl) havevery poor activity against these bacteria. This is in marked contrast totheir respective activities against M. tuberculosis, where the 4-halocompounds generally display very good activity but the tricyclicbenzoxaboroles without a 4-halogen are poorer (compare the M.tuberculosis MIC values for the same set of compounds in Tables 4A and4B).

Table 3 provides MIC Values Against non-Mycobacterial strains forCompounds of Formula II or Formula IIa

MIC: A. MIC: E. baumannii MIC: P. MIC: E. coli K12 ATCC aeruginosa coliK12 tolC::Tn10 17978 PA01 Compound [ug/mL] [ug/mL] [ug/mL] [ug/mL]Example 1 >64 >64 >64 >64 G1-Br Example 2  64  64  64  64 G2-Br Example3 >64 >64 >64 >64 G3-Cl Example 4  64  64  64  64 G4-Cl Example 5  32 64 >64  4 G5-F Example 6 — — — — G6-I Example 7 >64 >64 >64 >64 G7-ClExample 8 >64 >64 >64 >64 G8-Br Example 9 >64 >64 >64 >64 G9-Br Example10 >64 >64 >64 >64 G10-Br Example 11 >64 >64 >64 >64 G11-Cl Example12 >64 >64 >64 >64 G12-Cl C1-H — — — — C2-H  2  4  2  2 C3-H — — — —C4-Br  64  64  64  64 C5-H — — — — C6-Cl  64  64  64  64 C7-Cl2 — — — —C8-Cl — — — — C9-Cl — — — — C10-H — — — — C11-H  2  2  4  2 C12-H  4  2 4  16 C13-Cl — — — — C14-Cl2 — — — — C15-F — — — — C16-Cl — — — —GSK3309930A AN12471.01 C17-Cl — — — — GSK3309934A AN12470.01 C18-Br — —— — GSK3337512A AN12344.01 C19-Br — — — — GSK3309932A AN12343.01

Example 21 LeuRS Expression and Purification

For biochemical analyses an N-terminal six histidine-tagged LeuRS wasover-expressed in Escherichia coli which were E. coli codon-optimised(GenScript, Piscataway N.J., USA), from human mitochondria andcytoplasm, and M. tuberculosis. N-terminal six histidine-tagged LeuRSproteins were over-expressed and purified according to Novagen (Madison,Wis., USA) using an E. coli BL21(DE3) T7 RNA polymerase over-expressionstrain.

Example 22 Aminoacylation Assay

Experiments were performed in 96-well microtiter plates, using 80 μLreaction mixtures containing 50 mM HEPES-KOH (pH 8.0), 30 mM MgCl₂ 30 mMKCl, 13 μM L-[¹⁴C]leucine (306 mCi/mmol, Perkin-Elmer), 15 uM total E.coli tRNA (Roche, Switzerland), 0.02% (w/v) BSA, 1 mM DTT, 0.2 μM LeuRSand 4 mM ATP at 30° C. Reactions were started by the addition of 4 mMATP. After 7 minutes, reactions were quenched and tRNA was precipitatedby the addition of 50 μL of 10% (w/v) TCA and transferred to 96-wellnitrocellulose membrane filter plates (Millipore Multiscreen HTS,MSHAN4B50). Each well was then washed three times with 100 μL of 5% TCA.Filter plates were then dried under a heat lamp and the precipitatedL-[¹⁴C]leucine tRNALeu were quantified by liquid scintillation countingusing a Wallac MicroBeta Trilux model 1450 liquid scintillation counter(PerkinElmer, Waltham, Mass., USA). The only difference was with thehuman cytoplasmic LeuRS when we used tRNA isolated from Brewer's Yeast(Roche Diagnostics GmbH).

Example 23 IC₅₀ Determination

To determine the inhibitor concentration, which reduces enzyme activityby 50% (IC₅₀), increasing concentrations of compound (AnacorPharmaceuticals Inc., Palo Alto, Calif., USA) were incubated with LeuRSenzyme, tRNA and L-leucine 20 minutes. Reactions were initiated by theaddition of 4 mM ATP. Reactions were stopped after 7 minutes thenprecipitated and counted to quantify radioactivity. IC50 values weredetermined using the Graphpad Prism software package (Graphpad SoftwareInc. (La Jolla, Calif., USA).

Example 24 HepG2 Cytotoxicity Assay

HepG2 cells (HB-8065) were fed fresh medium (Essential Minimum EagleMedium, EMEM, supplemented with 5% fetal calf serum and 2mM L-glutamine)the day before subculturing the plates. On the day of plate seeding, acell suspension of 100,000 cells/mL in culture medium was prepared. Cellsuspension (100 uL) was added in each well of a black 96-well microplatewith clear bottom, collagen coated, (Becton Dickinson) except in column11, that was dispensed only 100 uL of culture medium. The plates wereincubated for 24 h. It was made up a range of 10 doses of testsubstances by preparing serial dilutions 1:2 from the stock solution in100% DMSO and made a dilution of 1:200 of each dose in medium, toachieve a final concentration of 0.5% of DMSO. After 24 h, culturemedium was removed from the plate and 150 uL of test compound dilutionswere added in two replicates and 150 uL of 0.5% DMSO in culture mediumto columns 11 and 12 (blank control). Plates were incubated for 48 andat 37° C., 5% CO2, 95% relative humidity. The medium was then removedand 200 uL of fresh culture medium was added and 50 uL of Resazurinsolution to each well and incubated for 1 h and a half. Plates wereremoved from incubator to allow the fluorescence to stabilise at roomtemperature protected from light for 15 min. For read out of viabilityof cells we used Resazurine (BDH). Resazurin is used as anoxidation-reduction indicator that yields a colorimetric change and afluorescent signal in response to metabolic activity. As cell grows,metabolic activity results in a chemical reduction of Resazurinindicated by a change from non-fluorescent blue to the reducedfluorescent pink form. The degree of Resazurin fluorescence istherefore, an indicator of the number of viable cells in the culturesystem. Fluorescence was measured at an excitation wavelength of 515 nmand an emission wavelength of 590 nm in a Microplate reader1420Multilabel HTS counter, Victor 2, (Wallac).

The fluorescence value of each well is corrected by subtracting thebackground value (average of column 11) from the absolute value. Thepercentages of inhibition are calculated relatively to the DMSO controlwells (average of column 12). For each compound, the average value ofthe duplicate samples is calculated and the curve is fitted to Sigmoidaldose-response (variable slope) nonlinear regression curve adjustment(GraphPad) in order to calculate the IC50 (Tox50).

Example 25 The Effect of Compounds Described Herein AgainstMycobacterium tuberculosis

Compounds of the present invention were tested for antibacterialactivity against a Mycobacterium tuberculosis species and also testedfor human liver cell toxicity using HepG2 cells. Exemplary compounds ofthe invention were compared to comparator compounds C1-H through C19-Br,as shown in Tables 4A and 4B.

Table 4A provides LeuRS inhibition IC50 values, MIC values against theM. tuberculosis standard strain Mtb H37Rv, toxicity values against humanHepG2 cells, and selectivity values for Certain Comparator TricyclicBenzoxaborole Compounds

HepG2 Human Human Mtb cell Mtb cyto mito H37Rv 48 h Selecti- LeuRS LeuRSLeuRS MIC Tox50 vity Compound Compound IC₅₀ IC₅₀ IC₅₀ (μM) (μM) IndexDesignation Structure (uM) (μM) (μM) (B) (A) (A/B) C1-H

   12.2 101 —  31 — — C2-H (racemic)

   0.506 272 >300    1.88 >50  >26 C3-H

   17.6  35.7 —   62 >50  >0.8 C4-Br

   0.07  31, (73,  67) >300    0.1   32   320 C5-H

   0.111  25.6 >300    0.6    1.8    3 C6-Cl

   0.05  38.8 >300    0.1   36.3   363 C7-Cl₂

   7.97 — —    2.5 >50  >20 C8-Cl

   6.05 — —  >5.0 >50    10 C9-Cl

   37.59 — —    5.0 >50  >10 C10-H

>300 — —  >5.0 >50    10 C11-H

   0.51 — —    1.56 >50 (40%)  >32 C12-H

   1.33 — —  >5.0   24.5  >4.9 C13-Cl

   2.16    5.0 >50  >10 C14-Cl₂

   4.67  >5.0 >50  >10 EXAMPLE 13 C15-F ((2S,8R)-2- (aminomethyl)-3-fluoro-7,8- dihydro-2H- 1,6,9-trioxa-9a- borabenzo[cd] azulen-8-yl)methanol

   0.48 — —    0.55 >50  >10 EXAMPLE 14 C16-Cl ((8R)-2- (aminomethyl)-3-chloro-8- methyl-7,8- dihydro-2H- 1,6,9-trioxa-9a- borabenzo[cd]azulen-8- yl)methanol

   4.17 — —    1.25 >50  >4 EXAMPLE 15 C17-Cl ((8S)-2- (aminomethyl)-3-chloro-8- methyl-7,8- dihydro-2H- 1,6,9-trioxa-9a- borabenzo[cd]azulen-8- yl)methanol

   3.13 — —    0.93 >50  >50 EXAMPLE 16 C18-Br ((8R)-2- (aminomethyl)-3-bromo-8- methyl-7,8- dihydro-2H- 1,6,9-trioxa-9a- borabenzo[cd]azulen-8- yl)methanol

   2.69 — —    1.25 >50  >40 EXAMPLE 17 C19-Br ((8S)-2- (aminomethyl)-3-bromo-8- methyl-7,8- dihydro-2H- 1,6,9-trioxa-9a- borabenzo[cd]azulen-8- yl)methanol

   1.97 — —    0.925 >50  >50

Table 4B provides LeuRS inhibition IC50 values, MIC values against theM. tuberculosis standard strain Mtb H37Rv, toxicity values against humanHepG2 cells, and selectivity values for Compounds of Formula II orFormula IIa

Human Human Mtb HepG2 Mtb cyto mito H37Rv cell 48 h LeuRS LeuRS LeuRSMIC Tox50 Selectivity Compound Compound IC₅₀ IC₅₀ IC₅₀ (μM) (μM) IndexDesignation Structure (uM) (μM) (μM) (B) (A) (A/B) EXAMPLE 1 G1-Br

0.154 0.18  >50  >277 EXAMPLE 2 G2-Br

0.115   118 >300 0.07    292.4    4177 EXAMPLE 3 G3-Cl

0.244 0.47  >50  >106 EXAMPLE 4 G4-Cl

0.148    94.7 >300 0.08 >1000 >12500 EXAMPLE 5 G5-F

0.46 0.6    49.1    164 EXAMPLE 6 G6-I

0.33 0.3    36.4    121 EXAMPLE 7 G7-Cl

1.08 >300 — 0.20  >50  >250 EXAMPLE 8 G8-Br

1.43 >300 — 0.30  >50  >167 EXAMPLE 9 G9-Br

1.25 >300 — 0.30  >50  >167 EXAMPLE 10 G10-Br

1.13 >300 0.16    460    2875 EXAMPLE 11 G11-Cl

0.68 >300 — 0.27  >50  >185 EXAMPLE 12 G25-Cl

0.78 >300 0.08    322    4025

As can be seen in Table 4B, for Examples 2, 4, 10 and 12 (G2-Br, G4-Cl,G10-Br and G12-Cl) there appears to be increased selectivity forinhibiting growth of M. tuberculosis versus toxicity for human HepG2cells for a tricyclic benzoxaborole compound with the third ring being aseven-membered ring between the 1 and 7 positions of the benzoxaborole,additionally having 4-halo, 3-aminomethyl substitution with (S)stereochemistry at the 3 position.

Tables 4A and 4B show a comparison of certain tricyclic benzoxaborolecompounds with and without halogen substitution, certain tricyclicbenzoxaborole compounds with and without halogen substitution atposition 4 of the benzoxaborole ring structure, and certain bicycliccompounds. From the Mtb H37Rv MIC values (B), and the HepG2 cell 48 hTox₅₀ values (A), it is possible to determine selectivity for inhibitionof M. tuberculosis versus inhibition (toxicity) of human cells for thesecompounds (see far right column of Tables 4A and 4B).

Compounds Example 2 G2-Br and Example 4 G4-Cl were found to haveselectivity indices against M. tuberculosis of 4177 and >12,500,respectively (see Table 4B). Further, as seen in Table 4B the IC₅₀values for these compounds against M. tuberculosis were found to besub-micromolar, at 0.13 and 0.1, respectively. As can be seen, theselectivity index (SI) of Example 2 G2-Br and Example 4 G4-Cl against M.tuberculosis is unexpectedly improved over other benzoxaborolecompounds. Example 2 G2-Br and Example 4 G4-Cl, which are tricyclicbenzoxaborole compounds having a halogen substituent at the C-4 positionof the benzoxaborole ring and an aminomethyl substituent at position C3of the benzoxaborole ring having “(S)” relative stereochemistry at thatstereocenter, are surprisingly more selective for activity against M.tuberculosis than other benzoxaborole compounds lacking some of thesefeatures versus inhibition (toxicity) of human cells for thesecompounds. In addition, the MIC values against M. tuberculosis H37Rvstrain for Example. 2 G2-Br and Example 4 G4-Cl are both <0.1 μM incontrast to other benzoxaborole compounds in this study.

Thus, as seen in Table 4B, compounds Example 2 G2-Br and Example 4 G4-Clwere found to have a SI against Mycobacterium tuberculosis of 4177(Example 2 G2-Br) and >12,500 (Example 4 G4-Cl), respectively. These SIvalues are surprisingly better than any of the comparator compoundstested to date.

Addition of a chloro or bromo substituent at C4 of the benzoxaborolering confers an unexpected increase in the selectivity index. C2-H(racemic; no halogen substituent at C4 of the benzoxaborole ring) has aselectivity index of >26 whereas Example 1 G1-Br (racemic; bromosubstituent at C-4 of the benzoxaborole ring) has an SI of >277.Similarly, Example 3 G3-Cl (racemic; chloro substituent at C-4 of thebenzoxaborole ring) has an SI of >106 compared to C2-H with an SI of>26.

Formation of a third ring involving the 1 and 7 positions of thebenzoxaborole ring confers an unexpected increase in the selectivityindex. C4-Br, the (S) enantiomer of a non-tricyclic benzoxaborolecomparator compound with a Br at the C4 position of the benzoxaborolering, has an SI of 320, whereas Example 2 G2-Br, the (S) enantiomer of atricyclic benzoxaborole with a Br at the C-4 position, has an SI of4177. Similarly, C6-Cl, the (S) enantiomer of a non-tricyclicbenzoxaborole comparator compound with a Cl at the C4 position of thebenzoxaborole ring, has an SI of 363, whereas Example 4 G4-Cl, the (S)enantiomer of a tricyclic benzoxaborole with a Cl at the C-4 position,has an SI of >12,500.

If one compares the SI of Example 2 G2-Br and Example 4 G4-Cl to the SIof C5-H, the (S) enantiomer of a non-tricyclic benzoxaborole comparatorcompound with a H at the C4 position of the benzoxaborole ring, one cansee the SI of such a compound without a halogen substituent at C4 isonly 3, indicating such a compound has very little selectivity forinhibiting M. tuberculosis compared to killing human cells.

Certain substitutions of the 7-membered tricyclic ring confer anunexpected increase in the selectivity index. Table 4B shows Example 9G9-Br and Example 11 G11-Cl with SI indices of >167 and >185,respectively, whereas comparator compounds C9-Cl (a tricyclicbenzoxaborole with a chloro substituent at C4 and —CH₃ substitution atR³ and R⁴ of the 7-membered ring) and C10-H (a tricyclic benzoxaborolewith a hydrogen at C4 and —CH₃ substitution at R³ and R⁴ of the7-membered ring) have SI indices of 10. This arguably indicates thatsubstitution at the R³ and R⁴ positions is not favored for selectivityfor M. tuberculosis versus inhibition (toxicity) of human cells forthese compounds. It also suggests that the presence of a halogen atposition C4 of the benzoxaborole ring (see C9-Cl) is not sufficient toovercome the negative effect of methyl substitution at both R³ and R⁴ ofthe 7-membered tricyclic ring at the R³/R⁴ position.

In other respects Example 2 G2-Br and Example 4 G4-Cl also have SIvalues unexpectedly higher than related open ring benzoxaboroles(substituted benzoxaboroles) lacking a halogen substituent at the C4position of the benzoxaborole ring. Compare the SI for C5-H (5) to theSIs for Example 2 G2-Br and Example 4 G4-Cl. Benzoxaboroles that are nottricyclic benzoxaboroles but which have a halogen at the C4 position ofthe benzoxaborole ring show improved SIs relative to no halogen, butstill exhibit SI values significantly lower than the SIs for Example 2G2-Br and Example 4 G4-Cl (compare C5-H to C3-Br and C6-Cl; but thencompare all three C5-H, C3-Br and C6-Cl to the SI values of Example 2G2-Br and Example 4 G4-Cl).

Thus, the tricyclic benzoxaboroles of the invention, particularlyExample 2 G2-Br and Example 4 G4-Cl, show surprisingly higher SIsrelative to the SIs of related benzoxaboroles for M. tuberculosis versushuman cells.

It is to be understood that the invention covers all combinations ofaspects with all other suitable aspects and/or exemplary embodimentsdescribed herein. It is to be understood that the invention also coversall combinations of exemplary embodiments with all other suitableaspects and/or exemplary embodiments described herein.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A compound having a structure as shown in Formula II:

wherein X is selected from chloro or bromo and R¹ and R² are each independently selected from H and —CH₃; or a pharmaceutically acceptable salt thereof. 2-21. (canceled) 